Sunscreen-wound healing compositions and methods for preparing and using same

ABSTRACT

The present invention pertains to therapeutic sunscreen-wound healing compositions useful to minimize and treat sunburn damage. The compositions comprise a therapeutically effective amount of (1) a sunscreen agent; (2) an anti-inflammatory; and, (3) a wound healing composition. In one embodiment the wound healing composition comprises (a) pyruvate; (b) an antioxidant; and (c) a mixture of saturated and unsaturated fatty acids. The therapeutic sunscreen-wound healing compositions may be utilized in a wide variety of pharmaceutical products. This invention also relates to methods for preparing and using the therapeutic sunscreen-wound healing compositions and the pharmaceutical products in which the therapeutic compositions may be used.

REFERENCE TO RELATED UNITED STATES APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 08/350,918filed Dec. 7, 1994 abandoned; which was a continuation-in-part of U.S.application Ser. No. 08/053,922 filed Apr. 26, 1993 abandoned; which wasa continuation of U.S. application Ser. No. 07/663,500 filed Mar. 1,1991 abandoned.

BACKGROUND OF INVENTION

1. Field of the Invention

This invention pertains to therapeutic sunscreen-wound healingcompositions useful to minimize and treat sunburn damage. Moreparticularly, the sunscreen-wound healing compositions comprise asunscreen agent, an anti-inflammatory agent, and a therapeutic woundhealing composition and/or its metabolites. This invention also pertainsto methods for preparing and using the therapeutic sunscreen-woundhealing compositions and the pharmaceutical products in which thetherapeutic compositions may be used.

A preferred embodiment of the therapeutic wound healing composition ofthis invention comprises (a) pyruvate selected from the group consistingof pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, andmixtures thereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acid wherein the fatty acids are those fatty acidrequired for the repair of cellular membranes and resuscitation ofmammalian cells.

2. Description of the Background Wound Healing

Wounds are internal or external bodily injuries or lesions caused byphysical means, such as mechanical, chemical vital, bacterial, orthermal means, which disrupt the normal continuity of structures. Suchbodily injuries include contusions, wounds in which the skin isunbroken, incisions, wounds in which the skin is broken by a cuttinginstrument, and lacerations, wounds in which the skin is broken by adull or blunt instrument. Wounds may be caused by accidents or bysurgical procedures.

Wound healing consists of a series of processes whereby injured tissueis repaired, specialized tissue is regenerated, and new tissue isreorganized. Wound healing consists of three major phases: a) aninflammation phase (0-3 days), b) a cellular proliferation phase (3-12days), and (c) a remodeling phase (3 days.4 months).

During the inflammation phase, platelet aggregation and clotting form amatrix which traps plasma proteins and blood cells to induce the influxof various types of cells. During the cellular proliferation phase, newconnective or granulation tissue and blood vessels are formed. Duringthe remodeling phase, granulation tissue is replaced by a network ofcollagen and elastin fibers leading to the formation of scar tissue.

When cells are injured or killed as a result of a wound, a wound healingstep is desirable to resuscitate the injured cells and produce new cellsto replace the dead cells. The healing process requires the reversal ofcytotoxicity, the suppression of inflammation, and the stimulation ofcellular viability and proliferation. Wounds require low levels ofoxygen in the initial stages of healing to suppress oxidative damage andhigher levels of oxygen in the later stages of healing to promotecollagen formation by fibroblasts.

Mammalian cells are continuously exposed to activated oxygen speciessuch as superoxide (O₂ --), hydrogen peroxide (H₂ O₂), hydroxyl radical(OH.), and singlet oxygen (¹ O₂). In vivo, these reactive oxygenintermediates are generated by cells in response to aerobic metabolism,catabolism of drugs and other xenobiotics, ultraviolet and x-rayradiation, and the respiratory burst of phagocytic cells (such as whiteblood cells) to kill invading bacteria such as those introduced throughwounds. Hydrogen peroxide, for example, is produced during respirationof most living organisms especially by stressed and injured cells.

These active oxygen species can injure cells. An important example ofsuch damage is lipid peroxidation which involves the oxidativedegradation of unsaturated lipid. Lipid peroxidation is highlydetrimental to membrane structure and function and can cause numerouscytopathological effects. Cells defend against lipid peroxidation byproducing radical scavengers such as superoxide dismutase, catalase, andperoxidase. Injured cells have a decreased ability to produce radicalscavengers. Excess hydrogen peroxide can react with DNA to causebackbone breakage, produce mutations, and alter and liberate bases.Hydrogen peroxide can also react with pyrimidines to open the 5,6-double bond, which reaction inhibits the ability of pyrimidines tohydrogen bond to complementary bases, Hallaender et al. (1971). Suchoxidative biochemical injury can result in the loss of cellular membraneintegrity, reduced enzyme activity, changes in transport kinetics,changes in membrane lipid content, and leakage of potassium ions, aminoacid, and other cellular material.

Antioxidants have been shown to inhibit damage associated with activeoxygen species. For example, pyruvate and other Alpha-ketoacids havebeen reported to react rapidly and stoichiometrically with hydrogenperoxide to protect cells from cytolytic effects, O'Donnell-Tormey etal., J. Exp. Mead., 165, pp. 500-514 (1987).

U.S. Pat. Nos. 3,920,835, 3,984,556, and 3,988,470, all issued to VanScott et al., disclose methods for treating acne, dandruff, and palmarkeratosis, respectively, which consist of applying to the affected areaa topical composition comprising from about 1% to about 20% of a loweraliphatic compound containing from two to six carbon atoms selected fromthe group consisting of Alpha-hydroxyacids, Alpha-ketoacids and estersthereof, and 3-hydroxybutryic acid in a pharmaceutically acceptablecarrier. The aliphatic compounds include pyruvic acid and lactic acid.

U.S. Pat. Nos. 4,105,783 and 4,197,316, both issued to Yu et al.,disclose a method and composition, respectively, for treating dry skinwhich consists of applying to the affected area a topical compositioncomprising from about 1% to about 20% of a compound selected from thegroup consisting of amides and ammonium salts of Alpha-hydroxyacids,β-hydroxyacids, and Alpha-ketoacids in a pharmaceutically acceptablecarrier. The compounds include the amides and ammonium salts of pyruvicacid and lactic acid.

U.S. Pat. No. 4,234,599, issued to Van Scott et at., discloses a methodfor treating actinic and nonactinic skin keratoses which consists ofapplying to the affected area a topical composition comprising aneffective amount of a compound selected from the group consisting ofAlpha-hydroxyacids, β-hydroxyacids, and Alpha-ketoacids in apharmaceutically acceptable carrier. The acidic compounds includepyruvic acid and lactic acid.

U.S. Pat. No. 4,294,852, issued to Wildnauer et al., discloses acomposition for treating skin which comprises the Alpha-hydroxyacids,β-hydroxyacids, and Alpha-ketoacids disclosed above by Van Scott et al.in combination with C₃ -C₈ aliphatic alcohols.

U.S. Pat. No. 4,663,166, issued to Veech, discloses an electrolytesolution which comprises a mixture of L-lactate and pyruvate in a ratiofrom 20:1 to 1:1, respectively, or a mixture of D-β-hydroxybutyrate andacetoacetate, in a ratio from 6:1 to 0.5:1, respectively.

Sodium pyruvate has been reported to reduce the number of erosions,ulcers, and hemorrhages on the gastric mucosa in guinea pigs and ratscaused by acetylsalicylic acid. The analgesic and antipyretic propertiesof acetylsalicylic acid were not impaired by sodium pyruvate, Puschmann,Arzneimittelforschung, 33, pp. 410-415 and 415-416 (1983).

Pyruvate has been reported to exert a positive inotropic effect instunned myocardium, which is a prolonged ventricular dysfunctionfollowing brief periods of coronary artery occlusions which does notproduce irreversible damage, Mentzer et al., Ann. Surg., 209, pp.629-633 (1989).

Pyruvate has been reported to produce a relative stabilization of leftventricular pressure and work parameter and to reduce the size ofinfarctions. Pyruvate improves resumption of spontaneous beating of theheart and restoration of normal rates and pressure development, Bungeret al., J. Mol. Cell. Cardiol., 18, pp. 423-438 (1986), Mochizuki etal., J. Physiol. (Paris), 76, pp. 805-812 (1980), Regitz et al.,Cardiovasc. Res., 15, pp. 652-658 (1981), Giannelli et al., Ann. Thorac.Surg., 21, pp. 386-396 (1976).

Sodium pyruvate has been report to act as an antagonist to cyanideintoxication (presumably through the formation of a cyanohydrin) and toprotect against the lethal effects of sodium sulfide and to retard theonset and development of functional, morphological, and biochemicalmeasures of acrylamide neuropathy of axons, Schwartz et al., Toxicol.Appl. Pharmacol., 50, pp. 437-442(1979), Sabri et al., Brain Res., 483,pp. 1-11 (1989).

A chemotherapeutic cure of advanced L1210 leukemia has been reportedusing sodium pyruvate to restore abnormally deformed red blood cells tonormal. The deformed red blood cells prevented adequate drug delivery totumor cells, Cohen, Cancer Chemother. Pharmacol., 5, pp. 175-179 (1981).

Primary cultures of heterotopic tracheal transplant exposed in vivo to7, 12-dimethyl-benz(a)anthracene were reported to be successfullymaintained in enrichment medium supplemented with sodium pyruvate alongwith cultures of interleukin-2 stimulated peripheral blood lymphocytes,and plasmacytomas and hybridomas, pig embryos, and human blastocysts,Shacter, J. Immunol. Methods, 99, pp. 259-270 (1987), Marchok et al.,Cancer Res., 37, pp. 1811-1821 (1977), Davis, J. Reprod. Fertil. Suppl.,33, pp. 115-124 (1985), Okamoto et al., No To Shinkei, 38, pp. 593-598(1986), Cohen et al., J. In Vitro Fert. Embryo Transfer, 2, pp. 59-64(1985).

U.S. Pat. Nos. 4,158,057, 4,351,835, 4,415,576, and 4,645,764, allissued to Stanko, disclose methods for preventing the accumulation offat in the liver of a mammal due to the ingestion of alcohol, forcontrolling weight in a mammal, for inhibiting body fat while increasingprotein concentration in a mammal, and for controlling the deposition ofbody fat in a living being, respectively. The methods compriseadministering to the mammal a therapeutic mixture of pyruvate anddihydroxyacetone, and optionally riboflavin. U.S. Pat. No. 4,548,937,issued to Stanko, discloses a method for controlling the weight gain ofa mammal which comprises administering to the mammal a therapeuticallyeffective amount of pyruvate, and optionally riboflavin. U.S. Pat. No.4,812,479, issued to Stanko, discloses a method for controlling theweight gain of a mammal which comprises administering to the mammal atherapeutically effective amount of dihydroxyacetone, and optionallyriboflavin and pyruvate.

Rats fed a calcium-oxalate lithogenic diet including sodium pyruvatewere reported to develop fewer urinary calculi (stones) than controlrats not given sodium pyruvate, Ogawa et al., Hinyokika Kiyo, 32, pp.1341-1347 (1986).

U.S. Pat. No. 4,521,375, issued to Houlsby, discloses a method forsterilizing surfaces which come into contact with living tissue. Themethod comprises sterilizing the surface with aqueous hydrogen peroxideand then neutralizing the surface with pyruvic acid.

U.S. Pat. No. 4,416,982, issued to Tauda et al., discloses a method fordecomposing hydrogen peroxide by reacting the hydrogen peroxide with aphenol or aniline derivative in the presence of peroxidase.

U.S. Pat. No. 4,696,917, issued to Lindstrom et al., discloses an eyeirrigation solution which comprises Eagle's Minimum Essential Mediumwith Earle's salts, chondroitin sulfate, a buffer solution,2-mercaptoethanol, and a pyruvate. The irrigation solution mayoptionally contain ascorbic acid and Alpha-tocopherol. U.S. Pat. No.4,725,586, issued to Lindstrom et al., discloses an irrigation solutionwhich comprises a balanced salt solution, chondroitin sulfate, a buffersolution, 2-mercaptoethanol, sodium bicarbonate or dextrose, a pyruvate,a sodium phosphate buffer system, and cystine. The irrigation solutionmay optionally contain ascorbic acid and gamma-tocopherol.

U.S. Pat. No. 3,887,702 issued to Baldwin, discloses a composition fortreating fingernails and toenails which consists essentially of soybeanoil or sunflower oil in combination with Vitamin E.

U.S. Pat. No. 4,847,069, issued to Bissett et al., discloses aphotoprotective composition comprising (a) a sorbohydroxamic acid, (b)an anti-inflammatory agent selected from steroidal anti-inflammatoryagents and a natural anti-inflammatory agent, and (c) a topical carrier.Fatty acids may be present as an emollient. U.S. Pat. No. 4,847,071,issued to Bissell et al., discloses a photoprotective compositioncomprising (a) a tocopherol or tocopherol ester radical scavenger, (b)an anti-inflammatory agent selected from steroidal anti-inflammatoryagents and a natural anti-inflammatory agent, and (c) a topical carrier.U.S. Pat. No. 4,847,072, issued to Bissett et al., discloses a topicalcomposition comprising not more than 25% tocopherol sorbate in a topicalcarrier.

U.S. Pat. No. 4,533,637, issued to Yamane et al., discloses a culturemedium which comprises a carbon source, a nucleic acid source precursor,amino acids, vitamins, minerals, a lipophilic nutrient, and serunalbumin, and cyclodextrins. The lipophilic substances includeunsaturated fatty acids and lipophilic vitamins such as Vitamin A, D,and E. Ascorbic acid may also be present.

United Kingdom patent application no. 2,196,348A, to Kovar et al.,discloses a synthetic culture medium which comprises inorganic salts,monosaccharides, amino acids, vitamins, buffering agents, and optionallysodium pyruvate adding magnesium hydroxide or magnesium oxide to theemulsion. The oil phase may include chicken fat.

U.S. Pat. No. 4,284,630, issued to Yu et al., discloses a method forstabilizing a water-in-oil emulsion which comprises adding magnesiumhydroxide or magnesium oxide to the emulsion. The oil phase may includechicken fat.

Preparation H™ has been reported to increase the rate of wound healingin artificially created rectal ulcers. The active ingredients inPreparation H™ are skin respiratory factor and shark liver oil,Subramanyam et al., Digestive Diseases and Sciences, 29, pp. 829-832(1984).

The addition of sodium pyruvate to bacterial and yeast systems has beenreported to inhibit hydrogen peroxide production, enhance growth, andprotect the systems against the toxicity of reactive oxygenintermediates. The unsaturated fatty acids and saturated fatty acidscontained within chicken fat enhanced membrane repair and reducedcytotoxicity. The antioxidants glutathione and thioglycollate reducedthe injury induced by oxygen radical species, Martin, Ph. D. thesis,(1987-89).

U.S. Pat. No. 4,615,697, issued to Robinson, discloses a controlledrelease treatment composition comprising a treating agent and abioadhesive agent comprising a water-swellable but water-insoluble,fibrous cross-linked carboxy-functional polymer.

European patent application no. 0410696A1, to Kellaway et al., disclosesa mucoadhesive delivery system comprising a treating agent and apolyacrylic acid cross-linked with from about 1% to about 20% by weightof a polyhydroxy compound such as a sugar, cyclitol, or lower polyhydricalcohol.

Inflammation

Inflammation is a nonspecific response caused by several kind of injury,including penetration of the host by infectious agents. Thedistinguishing feature of inflammation is dilation and increasedpermeability of minute blood vessels. Direct injury, such as that causedby toxins elaborated by microorganisms, leads to destruction of vascularendothelium and increased permeability to plasma proteins, especially inthe venules and venular capillaries. Mediators of secondary injury areliberated from the site of direct injury. As a result, gaps form betweenvascular endothelial cells through which plasma proteins escape.Granulocytes, monocytes, and erythrocytes may also leave vascularchannels. Mediators of secondary injury include unknown substances andhistamine, peptides (kinins), kinin-forming enzymes (kininogenases), anda globulin permeability factor. These mediators are blocked from actionby antihistamines and sympathoamines, and are most pronounced in effecton venules, although lymphvascular endothelium also becomes more porousas a part of secondary injury.

The beneficial effect of the inflammatory response is the production of:(1) leukocytes in great numbers; (2) plasma proteins, nonspecific andspecific humoral agents, fibrinogen that on conversion to fibrin aid inlocalization of the infectious process while acting as a matrix forphagocytosis; and (3) increased blood and lymph flow that dilutes andflushes toxic materials while causing a local increase in temperature.

In the early stages of inflammation, the exudate is alkaline andneutrophilic polymorphonuclear leukocytes predominate. As lactic acidaccumulates, presumably from glycolysis, the Ph drops and macrophagesbecome the predominant cell type. Lactic acid and antibodies in theinflammatory exudate may inhibit parasites, but the majoranti-infectious effect of the inflammatory response is attributable tophagocytic cells.

The inflammatory response consists of three successive phases: (a)increased vascular permeability with resulting edema and swelling, (b)cellular infiltration and phagocytoses, and (c) proliferation of thefibroblasts, synthesizing new connective tissue to repair the injury. Alarge number of so-called mediators of inflammation have been implicatedin the inflammatory process primarily in terms of their capacity toinduce vasodilation and increase permeability.

The initial increase in capillary permeability and vasodilation in aninflamed joint is followed by an increase in metabolism of the jointtissues. Leakage of fibrinogen into the wound, where proteolytic enzymesconvert it into fibrin, establishes a capillary and lymphatic blockade.The concentrations of components of the ground substance of connectivetissue collagen, mucopolysaccharides, glycoproteins, and nonfibrousproteins are greatly increased during this process. As the exudativephase of the inflammation subsides, the fibroblast is found to be thedominant cell in the wounded zone. It first proliferates, thensynthesizes extracellular material, including new collagen fibers andacid mucopolysaccharides, which are laid down to form the new tissuematrix.

The inflammatory phenomenon includes fenestration of themicrovasculature, leakage of the elements of blood into the interstitialspaces, and migration of leukocytes into the inflamed tissue. On amacroscopic level, this is usually accompanied by the familiar clinicalsigns of erythema, edema tenderness (hyperalgesia), and pain. Duringthis complex response, chemical mediators such as histamine,5-hydroxytryptamine (5-HT), slow-reacting substance of anaphylaxis(SRS-A), various chemotactic factors, bradykinin, and prostaglandins areliberated locally. Phagocytic cells migrate into the area, and cellularlysosomal membranes may be raptured, releasing lytic enzymes. All theseevents may contribute to the inflammatory response.

Sunburn

Sunburn is an acute reaction by the skin to excessive exposure tosunlight. Ordinary sunburn results from overexposure of the skin toultraviolet waves of about 3000 Angstroms. Sunburn symptoms appear in 1to 24 hours and, except in severe reactions, pass their peak in 72hours. Following exposure to sunlight, the epidermis thickens and themelanocytes produce melanin at an increased rate, providing some naturalprotection against further exposure. Skin changes range from a milderythema with subsequent evanescent scaling, to pain, swelling, skintenderness, and blisters from more prolonged exposure. Fever, chills,weakness, and shock may appear if a large portion of the body surface isaffected. Secondary infection, particularly furunculosis, is the mostcommon late complication. The skin may remain hypervulnerable tosunlight for one to several weeks when pronounced exfoliation hasoccurred.

Sunscreen agents are very effective for preventing sunburn. A usefulsunscreen agent formulation is 5% para-aminobenzoic acid (PABA) in ethylalcohol or in a gel. The esters of para-aminobenzoic acid in an alcoholbase are only slightly less effective. Patients who do not toleratepara-aminobenzoic acid or its esters may use a benzophenone or titaniumdioxide sunscreen agent. Early treatment of extensive and severe sunburnwith a topical or systemic corticosteroid decreases discomfortconsiderably.

Chronic exposure to sunlight has an aging effect on the skin. Wrinklingand elastosis (yellow discoloration with small yellow nodules) are themost common consequences of long-term exposure. Precancerous keratoticlesions (actinic keratoses) are a frequent, disturbing consequence ofyears of overexposure. Squamous and basal cell carcinoma of the skin areespecially common in people who work outdoors.

SUMMARY OF THE INVENTION

This invention pertains to therapeutic sunscreen-wound healingcompositions useful to minimize and treat sunburn damage. Thecompositions of this invention comprise a therapeutically effectiveamount of (1) a sunscreen agent; (2) an anti-inflammatory agent; and,(3) a wound healing composition. A preferred embodiment of the woundhealing composition of this invention comprises (a) pyruvate selectedfrom the group consisting of pyruvic acid, pharmaceutically acceptablesalts of pyruvic acid, and mixtures thereof; (b) an antioxidant; and (c)a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells. The therapeuticsunscreen-wound healing compositions of this invention may be utilizedin a wide variety of pharmaceutical products. This invention alsorelates to methods for preparing and using the therapeuticsunscreen-wound healing compositions and the pharmaceutical products inwhich the therapeutic compositions may be used.

This invention further comprises augmented therapeutic sunscreen-woundhealing compositions comprising (1) a sunscreen agent; (2) ananti-inflammatory agent; and, (3) a therapeutic wound healingcomposition in combination with one or more additional medicaments. Thisinvention also relates to methods for preparing and using the augmentedtherapeutic antikeratolytic-wound healing compositions and thepharmaceutical products in which the augmented compositions may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts in bar graph format the viability of U937 monocytic cellsfollowing exposure of the cells to various antioxidants (Examples 1-5).

FIG. 2 depicts in bar graph format the viability of U937 monocytic cellsfollowing exposure of the cells to various combinations of antioxidants(Examples 6-13).

FIG. 3 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells following exposure of the cells tovarious antioxidants (Examples 14-18).

FIG. 4 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells following exposure of the cells tovarious combinations of antioxidants (Examples 19-26).

FIG. 5 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells following exposure of the cells tovarious combinations of antioxidants with and without a mixture ofsaturated and unsaturated fatty acids (Examples 27-32).

FIG. 6 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes following exposure of the cells tovarious antioxidants with and without a mixture of saturated andunsaturated fatty acids (Examples 33-42).

FIG. 7 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes following exposure of the cells tovarious combinations of antioxidants with and without a mixture ofsaturated and unsaturated fatty acids (Examples 43-52).

FIG. 8 depicts in bar graph format a summary analysis of the levels ofhydrogen peroxide produced by epidermal keratinocytes following exposureof the cells to the individual components of the wound healingcomposition, to various combinations of the wound healing composition,and to the wound healing composition.

FIGS. 9A-9D are photographs of wounded mice after 4 days of treatmentwith: no composition (FIG. 9A, control); a petrolatum base formulationcontaining live yeast cell derivative, shark oil, and a mixture ofsodium pyruvate, vitamin E, and chicken fat (FIG. 9B); a petrolatum baseformulation containing live yeast cell derivative and shark oil (FIG.9C); and Preparation H™ (FIG. 9D).

FIG. 10 is a photograph of a wounded mouse after 4 days of treatmentwith a petrolatum base formulation only (Example D).

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to therapeutic sunscreen-wound healingcompositions which comprise (1) a sunscreen agent; (2) ananti-inflammatory; and, (3) a wound healing composition and/or itsmetabolites. In one embodiment the wound healing composition comprises(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;(b) an antioxidant; and (c) a mixture of saturated and unsaturated fattyacids wherein the fatty acids are those fatty acids required for therepair of cellular membranes and resuscitation of mammalian cells.

Applicant has discovered therapeutic wound healing compositions forpreventing and reducing injury to mammalian cells and increasing theresuscitation rate of injured mammalian cells. Cells treated with thetherapeutic wound healing compositions of the present invention showdecreased levels of hydrogen peroxide production, increased resistanceto cytotoxic agents, increased rates of proliferation, and increasedviability. Cellular cultures containing the therapeutic wound healingcompositions showed enhanced differentiation and proliferation overcontrol cultures and rapidly formed attachments or tight junctionsbetween the cells to form an epidermal sheet. Wounded mammals treatedwith the therapeutic wound healing compositions show significantlyimproved wound closing and healing over untreated mammals and mammalstreated with conventional healing compositions. The wound healingcompositions may be used alone or in combination with other medicaments.

The therapeutic wound healing compositions of this invention aredescribed as Embodiment One. There are several aspects of EmbodimentOne. In a first aspect, (I.A), the therapeutic wound healing compositioncomprises (a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells. In a second aspect of Embodiment One (I.B), thetherapeutic wound healing composition comprises (a) pyruvate selectedfrom the group consisting of pyruvic acid, pharmaceutically acceptablesalts of pyruvic acid, and mixtures thereof, (b) lactate selected fromthe group consisting of lactic acid, pharmaceutically acceptable saltsof lactic acid, and mixtures thereof, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells. In a third aspect of Embodiment One (I.C), thetherapeutic wound healing composition comprises (a) an antioxidant and(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells. In a fourth aspect ofEmbodiment One (I.D), the therapeutic wound healing compositioncomprises (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof,(b) an antioxidant, and (c) a mixture of saturated and unsaturated fattyacids wherein the fatty acid are those fatty acid required for therepair of cellular membranes and resuscitation of mammalian cells.

The therapeutic wound healing compositions of this invention are furthercombined with a therapeutically effective amount of (1) a sunscreenagent and (2) an anti-inflammatory (collectively referred to as "X") toform therapeutic sunscreen-wound healing compositions (IA-D+X). Thetherapeutic sunscreen-wound healing compositions may be used alone or incombination with other medicaments. This invention also pertains tomethods for preparing and using the sunscreen-wound healing compositionsand the pharmaceutical products in which the therapeutic compositionsmay be used.

The therapeutic sunscreen-wound healing compositions of this inventionmay be further combined with one or more additional medicaments fortreating wounds to form augmented sunscreen-wound healing compositions.This invention also relates to methods for preparing and using theaugmented therapeutic sunscreen-wound healing compositions and thepharmaceutical products in which the augmented compositions may be used.

The term "injured cell" as used herein means a cell that has anyactivity disrupted for any reason. For example, an injured cell may be acell that has injured membranes or damaged DNA, RNA, and ribosomes, forexample, a cell which has (a) injured membranes so that transportthrough the membranes is diminished resulting in an increase in toxinsand normal cellular wastes inside the cell and a decrease in nutrientsand other components necessary for cellular repair inside the cell, (b)an increase in concentration of oxygen radicals inside the cell becauseof the decreased ability of the cell to produce antioxidants andenzymes, or (c) damaged DNA, RNA, and ribosomes which must be repairedor replaced before normal cellular functions can be resumed. The term"resuscitation" of injured mammalian cells as used herein means thereversal of cytotoxicity, the stabilization of the cellular membrane, anincrease in the proliferation rate of the cell, and/or the normalizationof cellular functions such as the secretion of growth factors, hormones,and the like. The term "cytotoxicity" as used herein means a conditioncaused by a cytotoxic agent that injures the cell. Injured cells do notproliferate because injured cells expend all energy on cellular repair.Aiding cellular repair promotes cellular proliferation.

The term "prodrug", as used herein, refers to compounds which undergobiotransformation prior to exhibiting their pharmacological effects. Thechemical modification of drugs to overcome pharmaceutical problems hasalso been termed "drug latentiation." Drug latentiation is the chemicalmodification of a biologically active compound to form a new compoundwhich upon in vivo enzymatic attack will liberate the parent compound.The chemical alterations of the parent compound are such that the changein physicochemical properties will affect the absorption, distributionand enzymatic metabolism. The definition of drug latentiation has alsobeen extended to include nonenzymatic regeneration of the parentcompound. Regeneration takes place as a consequence of hydrolytic,dissociative, and other reactions not necessarily enzyme mediated. Theterms prodrugs, latentiated drugs, and bioreversible derivatives areused interchangeably. By inference, latentiation implies a time lagelement or time component involved in regenerating the bioactive parentmolecule in vivo. The term prodrug is general in that it includeslatentiated drug derivatives as well as those substances which areconverted after administration to the actual substance which combineswith receptors. The term prodrug is a generic term for agents whichundergo biotransformation prior to exhibiting their pharmacologicalactions. In the case where the administered drug is not the activeagent, but rather is biotransformed to the active agent, the term"prodrug" also includes compounds which may not necessarily undergobiotransformation to the administered drug but may undergobiotransformation to the active agent which exhibits the desiredpharmacological effect.

The term "metabolite", as used herein, refers to any substance producedby metabolism or by a metabolic process. "Metabolism", as used herein,refers to the various chemical reactions involved in the transformationof molecules or chemical compounds occurring in tissue and the cellstherein.

I. WOUND HEALING COMPOSITIONS

A. Embodiment One (LA-D)

The cells which may be related with the therapeutic wound healingcompositions in the present invention are mammalian cells. Althoughapplicant will describe the present therapeutic wound healingcompositions as useful for treating mammalian epidermal keratinocytesand mammalian monocytes, applicant contemplates that the therapeuticwound healing compositions may be used to protect or resuscitate allmammalian cells. Keratinocytes are representative of normal mammaliancells and are the fastest proliferating cells in the body. Thecorrelation between the reaction of keratinocytes to injury and therapyand that of mammalian cells in general is very high. Monocytes arerepresentative of specialized mammalian cells such as the white bloodcells in the immune system and the organ cells in liver, kidney, heart,and brain. The mammalian cells may be treated in vivo and in vitro.

Epidermal keratinocytes are the specialized epithelial cells of theepidermis which synthesize keratin, a scleroprotein which is theprincipal constituent of epidermis, hair, nails, horny tissue, and theorganic matrix of the enamel of teeth. Mammalian epidermal keratinocytesconstitute about 95% of the epidermal cells and together withmelanocytes form the binary system of the epidermis. In its varioussuccessive stages, epidermal keratinocytes are also known as basalcells, prickle cells, and granular cells.

Monocytes are mononuclear phagocytic leukocytes which undergorespiratory bursting and are involved in reactive oxygen mediated damagewithin the epidermis. Leukocytes are white blood cells or corpuscleswhich may be classified into two main groups: granular leukocytes(granulocytes) which are leukocytes with abundant granules in thecytoplasm and nongranular leukocytes (nongranulocytes) which areleukocytes without specific granules in the cytoplasm and which includethe lymphocytes and monocytes. Phagocyte cells are cells which ingestmicroorganisms or other cells and foreign particles. Monocytes are alsoknown as large mononuclear leukocytes, and hyaline or transitionalleukocytes.

Epidermal keratinocytic cells and monocytic cells have multiple oxygengenerating mechanisms and the degree to which each type of mechanismfunctions differs in each type of cell. In monocytes, for example, therespiratory bursting process is more pronounced than in epidermalkeratinocytes. Hence, the components in the therapeutic wound healingcompositions of the present invention may vary depending upon the typesof cells involved in the condition being treated.

As set out above, in a first aspect of Embodiment One (I.A), thetherapeutic wound healing composition for treating mammalian cells,preferably epidermal keratinocytes, comprises (a) pyruvate selected fromthe group consisting of pyruvic acid, pharmaceutically acceptable saltsof pyruvic acid, and mixtures thereof, (b) an antioxidant, and (c) amixture of saturated and unsaturated fatty acids wherein the fatty acidsare those fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a second aspect of Embodiment One(I.B), the therapeutic wound healing composition for treating mammaliancells, preferably epidermal keratinocytes, comprises (a) pyruvateselected from the group consisting of pyruvic acid, pharmaceuticallyacceptable salts of pyruvic acid, and mixtures thereof, (b) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, and (c) a mixtureof saturated and unsaturated fatty acids wherein the fatty acids arethose fatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a third aspect of Embodiment One(I.C), the therapeutic wound healing composition for treating mammaliancells, preferably epidermal keratinocytes, comprises (a) an antioxidantand (b) a mixture of saturated and unsaturated fatty acids wherein thefatty acids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells. In a fourth aspect ofEmbodiment One (I.D), the therapeutic wound healing composition fortreating mammalian cells, preferably monocytes, comprises (a) lactateselected from the group consisting of lactic acid, pharmaceuticallyacceptable salts of lactic acid, and mixtures thereof, (b) anantioxidant, and (c) a mixture of saturated and unsaturated fatty acidswherein the fatty acids are those fatty acids required for the repair ofcellular membranes and resuscitation of mammalian cells.

Pyruvic acid (2-oxopropanoic acid,Alpha-ketopropionic acid, CH₃ COCOOH)or pyruvate is a fundamental intermediate in protein and carbohydratemetabolism and in the citric acid cycle. The citric acid cycle(tricarboxylic acid cycle, Kreb's cycle) is the major reaction sequencewhich executes the reduction of oxygen to generate adenosinetriphosphate (ATP) by oxidizing organic compounds in respiring tissuesto provide electrons to the transport system. Acetyl coenzyme A ("activeacetyl") is oxidized in this process and is thereafter utilized in avariety of biological processes and is a precursor in the biosynthesisof many fatty acids and sterols. The two major sources of acetylcoenzyme A are derived from the metabolism of glucose and fatty acids.Glycolysis consists of a series of transformations wherein each glucosemolecule is transformed in the cellular cytoplasm into two molecules ofpyruvic acid. Pyruvic acid may then enter the mitochondria where it isoxidized by coenzyme A in the presence of enzymes and cofactors toacetyl coenzyme A. Acetyl coenzyme A can then enter the citric acidcycle.

In muscle, pyruvic acid (derived from glycogen) can be reduced to lacticacid during anerobic metabolism which can occur during exercise. Lacticacid is reoxidized and partially retransformed to glycogen during rest.Pyruvate can also act as an antioxidant to neutralize oxygen radicals inthe cell and can be used in the multifunction oxidase system to reversecytotoxicity.

The pyruvate in the present invention may be selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, prodrugs of pyruvic acid, and mixtures thereof. In general, thepharmaceutically acceptable salts of pyruvic acid may be alkali saltsand alkaline earth salts. Preferably, the pyruvate is selected from thegroup consisting of pyruvic acid, lithium pyruvate, sodium pyruvate,potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate,manganese pyruvate, methyl pyruvate, Alpha-ketoglutaric acid, andmixtures thereof. More preferably, the pyruvate is selected from thegroup of salts consisting of sodium pyruvate, potassium pyruvate,magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate,and the like, and mixtures thereof. Most preferably, the pyruvate issodium pyruvate.

The amount of pyruvate present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of pyruvate is that amount ofpyruvate necessary for the inventive composition to prevent and reduceinjury to mammalian cells or increase the resuscitation rate of injuredmammalian cells. The exact amount of pyruvate is a matter of preferencesubject to such factors as the type of condition being treated as wellas the other ingredients in the composition. In a preferred embodiment,pyruvate is present in the therapeutic wound healing composition in anamount from about 10% to about 50%, preferably from about 20% to about45%, and more preferably from about 25% to about 40%, by weight of thetherapeutic wound healing composition.

L-Lactic acid ((S)-2-hydroxypropanoic acid, (+) Alpha-hydroxypropionicacid, CH₃ CHOHCOOH) or lactate occurs in small quantities in the bloodand muscle fluid of mammals. Lactic acid concentration increases inmuscle and blood after vigorous activity. Lactate is a component in thecellular feedback mechanism and inhibits the natural respiratorybursting process of cells thereby suppressing the production of oxygenradicals.

The lactate in the present invention may be selected from the groupconsisting of lactic acid, pharmaceutically acceptable salts of lacticacid, prodrugs of lactic acid, and mixtures thereof. In general, thepharmaceutically acceptable salts of lactic acid may be alkali salts andalkaline earth salts. Preferably, the lactate is selected from the groupconsisting of lactic acid, lithium lactate, sodium lactate, potassiumlactate, magnesium lactate, calcium lactate, zinc lactate, manganeselactate, and the like, and mixtures thereof. More preferably, thelactate is selected from the group consisting of lactic acid, sodiumlactate, potassium lactate, magnesium lactate, calcium lactate, zinclactate, manganese lactate, and mixtures thereof. Most preferably, thelactate is lactic acid.

The amount of lactate present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of lactate is that amount oflactate necessary for the inventive composition to prevent and reduceinjury to mammalian cells or increase the resuscitation rate of injuredmammalian cells. For an ingestible composition, a therapeuticallyeffective amount of lactate is that amount necessary to suppress therespiratory bursting process of white blood cells to protect andresuscitate the mammalian cells. In general, a therapeutically effectiveamount of lactate in an ingestible composition is from about 5 to about10 times the amount of lactate normally found in serum. The exact amountof lactate is a matter of preference subject to such factors as the typeof condition being treated as well as the other ingredients in thecomposition. In a preferred embodiment, lactate is present in thetherapeutic wound healing composition in an amount from about 10% toabout 50%, preferably from about 20% to about 45%, and more preferablyfrom about 25% to about 40%, by weight of the therapeutic wound healingcomposition.

Antioxidants are substances which inhibit oxidation or suppressreactions promoted by oxygen or peroxides. Antioxidants, especiallylipid-soluble antioxidants, can be absorbed into the cellular membraneto neutralize oxygen radicals and thereby protect the membrane. Theantioxidants useful in the present invention may be selected from thegroup consisting of all forms of Vitamin A including retinol and3,4-didehydroretinol, all forms of carotene such as Alpha-carotene,β-carotene (beta, β-carotene), gamma-carotene, delta-carotene, all formsof Vitamin C (D-ascorbic acid, L-ascorbic acid), all forms of tocopherolsuch as Vitamin E (Alpha-tocopherol,3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-decyl)-2H-1-benzopyran-6-ol), β-tocopherol, gamma-tocopherol, delta-tocopherol,tocoquinone, tocotrienol, and Vitamin E esters which readily undergohydrolysis to Vitamin E such as Vitamin E acetate and Vitamin Esuccinate, and pharmaceutically acceptable Vitamin E salts such asVitamin E phosphate, prodrugs of Vitamin A, carotene, Vitamin C, andVitamin E, pharmaceutically acceptable salts of Vitamin A, carotene,Vitamin C, and Vitamin E, and the like, and mixtures thereof.Preferably, the antioxidant is selected from the group of lipid-solubleantioxidants consisting of Vitamin A, β-carotene, Vitamin E, Vitamin Eacetate, and mixtures thereof. More preferably, the antioxidant isVitamin E or Vitamin E acetate. Most preferably, the antioxidant isVitamin E acetate.

The amount of antioxidant present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of antioxidant is that amountof antioxidant necessary for the inventive composition to prevent andreduce injury to mammalian cells or increase the resuscitation rate ofinjured mammalian cells. The exact amount of antioxidant is a matter ofpreference subject to such factors as the type of condition beingtreated as well as the other ingredients in the composition. In apreferred embodiment, the antioxidant is present in the therapeuticwound healing composition in an amount from about 0.1% to about 40%,preferably from about 0.2% to about 30%, and more preferably from about0.5% to about 20%, by weight of the therapeutic wound healingcomposition.

The mixture of saturated and unsaturated fatty acids in the presentinvention are those fatty acid required for the repair of mammaliancellular membranes and the production of new cells. Fatty acids arecarboxylic acid compound found in animal and vegetable fat and oil.Fatty acids are classified as lipids and are composed of chains of alkylgroups containing from 4 to 22 carbon atoms and 0-3 double bonds andcharacterized by a terminal carboxyl group, --COOH. Fatty acids may besaturated or unsaturated and may be solid, semisolid, or liquid. Themost common saturated fatty acids are butyric acid (C₄), lauric acid(C₁₂), palmitic acid (C₁₆), and stearic acid (C₁₈). Unsaturated fattyacid are usually derived from vegetables and consist of alkyl chainscontaining from 16 to 22 carbon atoms and 0-3 double bond with thecharacteristic terminal carboxyl group. The most common unsaturatedfatty acids are oleic acid, linoleic acid, and linolenic acid (all C₁₈acids).

In general, the mixture of saturated and unsaturated fatty acid requiredfor the repair of mammalian cellular membranes in the present inventionmay be derived from animal and vegetable fats and waxes, prodrugs ofsaturated and unsaturated fatty acids useful in the present invention,and mixtures thereof. For example, the fatty acids in the therapeuticwound healing composition may be in the form of mono-, di-, ortrigylcerides, or free fatty acids, or mixtures thereof, which arereadily available for the repair of injured cells. Cells produce thechemical components and the energy required for cellular viability andstore excess energy in the form of fat. Fat is adipose tissue storedbetween organs of the body to furnish a reserve supply of energy. Thepreferred animal fats and waxes have a fatty acid composition similar tothat of human fat and the fat contained in human breast milk. Thepreferred animal fats and waxes may be selected from the groupconsisting of human fat, chicken fat, cow fat (defined herein as abovine domestic animal regardless of sex or age), sheep fat, horse fat,pig fat, and whale fat. The more preferred animal fats and waxes may beselected from the group consisting of human fat and chicken fat. Themost preferred animal fat is human fat. Mixtures of other fats andwaxes, such as vegetable waxes (especially sunflower oil), marine oils(especially shark liver oil), and synthetic waxes and oils, which have afatty acid composition similar to that of animal fats and waxes, andpreferably to that of human fats and waxes, may also be employed.

In a preferred embodiment, the mixture of saturated and unsaturatedfatty acids has a composition similar to that of human fat and comprisesthe following fatty acids: butyric acid, caproic acid, caprylic acid,capric acid, lauric acid, myristic acid, myristoleic acid, palmiticacid, palmitoleic acid, stearic, oleic acid, linoleic acid, linolenicacid, arachidic acid, and gadoleic acid. Preferably, butyric acid,caproic acid, caprylic acid, capric acid, lauric acid, myristic acid,myristoleic acid, palmitic acid, palmitoleic acid, stearic, oleic acid,linoleic acid, linolenic acid, arachidic acid, and gadoleic acid arepresent in the mixture in about the following percentages by weight,respectively (carbon chain number and number of unsaturations are shownparenthetically, respectively): 0.2%-0.4% (C₄), 0.1% (C₆), 0.3%-0.8%(C₈), 2.2%-3.5% (C₁₀), 0.9%-5.5% (C₁₂), 2.8%-8.5% (C₁₄), 0.1%-0.6%(C_(14:1)), 23.2%-24.6% (C₁₆), 1.8%-3.0% (C_(16:1)), 6.9%-9.9% (C₁₈),36.0%-36.5% (C_(18:1)) 20%-20.6% (C_(18:2)), 7.5-7.8% (C_(18:3)),1.1%-4.9% (C₂₀), and 3.3%-6.4% (C_(20:1)).

In another preferred embodiment, the mixture of saturated andunsaturated fatty acids is typically chicken fat comprising thefollowing fatty acids: lauric acid, myristic acid, myristoleic acid,pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid,margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid,arachidic acid, and gadoleic acid. Preferably, lauric acid, myristicacid, myristoleic acid, pentadecanoic acid, palmitic acid, palmitoleicacid, margaric acid, margaroleic acid, stearic, oleic acid, linoleicacid, linolenic acid, arachidic acid, and gadoleic acid are present inthe mixture in about the following percentages by weight, respectively:0.1% (C₁₂), 0.8% (C₁₄), 0.2% (C_(14:1)), 0.1% (C₁₅), 25.3% (C₁₆), 7.2%(C_(16:1)), 0.1% (C₁₇), 0.1% (C_(17:1)), 6.5% (C₁₈), 37.7% (C_(18:1)),20.6% (C_(18:2)), 0.8% (C_(18:3)), 0.2% (C₂₀), and 0.3% (C_(20:1)), allpercentages ±10%.

In another preferred embodiment, the mixture of saturated andunsaturated fatty acids comprises lecithin. Lecithin(phosphatidylcholine) is a phosphatide found in all living organisms(plants and animals) and is a significant constituent of nervous tissueand brain substance. Lecithin is a mixture of the diglycerides ofstearic, palmitic, and oleic acids, linked to the choline ester ofphosphoric acid. The product of commerce is predominantly soybeanlecithin obtained as a by-product in the manufacturing of soybean oil.Soybean lecithin contains palmitic acid 11.7%, stearic 4.0%, palmitoleic8.6%, oleic 9.8%, linoleic 55.0%, linolenic 4.0%, C₂₀ to C₂₂ acids(includes arachidonic) 5.5%. Lecithin may be represented by the formula:##STR1## wherein R is selected from the group consisting of stearic,palmitic, and oleic acid.

The above fatty acids and percentages thereof present in the fatty acidmixture are given as an example. The exact type of fatty acid present inthe fatty acid mixture and the exact amount of fatty acid employed inthe fatty acid mixture may be varied in order to obtain the resultdesired in the final product and such variations are now within thecapabilities of those skilled in the art without the need for undueexperimentation.

The mount of fatty acid present in the therapeutic wound healingcompositions of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of fatty acids is that amountof fatty acids necessary for the inventive composition to prevent andreduce injury to mammalian cells or increase the resuscitation rate ofinjured mammalian cells. The exact amount of fatty acids employed issubject to such factors as the type and distribution of fatty acidsemployed in the mixture, the type of condition being treated, and theother ingredients in the composition. In a preferred embodiment, thefatty acids are present in the therapeutic wound healing composition inan amount from about 10% to about 50%, preferably from about 20% toabout 45%, and more preferably from about 25% to about 40%, by weight ofthe therapeutic wound healing composition.

In accord with the present invention, the therapeutic wound healingcompositions of Embodiment One (I.A-D) for treating mammalian cells maybe selected from the group consisting of:

(I.A)(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.C)(a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D)(a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acid wherein the fattyacid are those fatty acid required for the repair of cellular membranesand resuscitation of mammalian cells.

Preferably, the wound healing compositions of Embodiment One (I) fortreating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B)(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.C)(a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

More preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.C)(a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

More preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes, may beselected from the group consisting of:

(I.A)(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(I.B)(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacid are those fatty acids required for the repair of cellular membranesand resuscitation of mammalian cells.

Most preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably epidermal keratinocytes,comprise:

(I.A)(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Most preferably, the wound healing compositions of Embodiment One (I)for treating mammalian cells, preferably monocytes, comprise:

(I.D)(a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells.

Throughout this disclosure, applicant will suggest various theories ormechanisms by which applicant believes the components in the therapeuticwound healing compositions and the antiviral agent function together inan unexpected synergistic manner to prevent and reduce injury tomammalian cells, increase the resuscitation rate of injured mammaliancells, and reduce vital titers. While applicant may offer variousmechanisms to explain the present invention, applicant does not wish tobe bound by theory. These theories are suggested to better understandthe present invention but are not intended to limit the effective scopeof the claims.

In the first aspect of Embodiment One (I.A), applicant believes thatpyruvate can be transported inside a cell where it can act as anantioxidant to neutralize oxygen radicals in the cell. Pyruvate can alsobe used inside the cell in the citric acid cycle to provide energy toincrease cellular viability, and as a precursor in the synthesis ofimportant biomolecules to promote cellular proliferation. In addition,pyruvate can be used in the multifunction oxidase system to reversecytotoxicity. Antioxidants, especially lipid-soluble antioxidants, canbe absorbed into the cell membrane to neutralize oxygen radicals andthereby protect the membrane. The saturated and unsaturated fatty acidsin the present invention are those fatty acids required for theresuscitation of mammalian cells and are readily available for therepair of injured cells and the proliferation of new cells. Cellsinjured by oxygen radicals need to produce unsaturated fatty acids torepair cellular membranes. However, the production of unsaturated fattyacids by cells requires oxygen. Thus, the injured cell needs high levelsof oxygen to produce unsaturated fatty acids and at the same time needsto reduce the level of oxygen within the cell to reduce oxidativeinjury. By providing the cell with the unsaturated fatty acids neededfor repair, the need of the cell for unsaturated fatty acids is reducedand the need for high oxygen levels is also reduced.

The combination of pyruvate inside the cell and an antioxidant in thecellular membrane functions in an unexpected synergistic manner toreduce hydrogen peroxide production in the cell to levels lower than canbe achieved by use of either type of component alone. The presence ofmixtures of saturated and unsaturated fatty acids in the therapeuticwound healing composition significantly enhances the ability of pyruvateand the antioxidant to inhibit reactive oxygen production. Bystabilizing the cellular membrane, unsaturated fatty acids also improvemembrane function and enhance pyruvate transport into the cell. Hence,the three components in the therapeutic wound healing composition of thefirst aspect of Embodiment One (I.A) function together in an unexpectedsynergistic manner to prevent and reduce injury to mammalian cells andincrease the resuscitation rate of injured mammalian cells.

In the second aspect of Embodiment One (I.B), lactate is employedinstead of an antioxidant. Antioxidants react with, and neutralize,oxygen radicals after the radicals are already formed. Lactate, on theother hand, is a component in the cellular feedback mechanism andinhibits the respiratory bursting process to suppress the production ofactive oxygen species. The combination of pyruvate to neutralize activeoxygen species and lactate to suppress the respiratory bursting processfunctions in a synergistic manner to reduce hydrogen peroxide productionin the cell to levels lower than can be achieved by use of either typeof component alone. The presence of mixtures of saturated andunsaturated fatty acids in the therapeutic wound healing compositionsignificantly enhances the ability of pyruvate and lactate to inhibitreactive oxygen production. Hence, the three components in thetherapeutic wound healing composition in the second aspect of EmbodimentOne (I.B) function together in a synergistic manner to protect andresuscitate mammalian cells.

In the third aspect of Embodiment One (I.C), the presence of mixtures ofsaturated and unsaturated fatty acids in the therapeutic wound healingcomposition in this embodiment significantly enhances the ability of theantioxidant to inhibit reactive oxygen production. The combination of anantioxidant to neutralize active oxygen species and fatty acids torebuild cellular membranes and reduce the need of the cell for oxygenfunctions in a synergistic manner to reduce hydrogen peroxide productionin the cell to levels lower than can be achieved by either type ofcomponent alone. Hence, the components in the therapeutic wound healingcomposition in the third aspect of Embodiment One (I.C) functiontogether in a synergistic manner to protect and resuscitate mammaliancells.

In the fourth aspect of Embodiment One (I.D), lactate is employedbecause the respiratory bursting process is more pronounced in monocytesthan in epidermal keratinocytes. The combination of lactate to suppressthe respiratory bursting process and an antioxidant to neutralize activeoxygen species functions in a synergistic manner to reduce hydrogenperoxide production in the cell to levels lower than can be achieved byeither component alone. The presence of mixtures of saturated andunsaturated fatty acids in the therapeutic wound healing composition inthis embodiment significantly enhances the ability of lactate and theantioxidant to inhibit reactive oxygen production. Hence, the threecomponents in the therapeutic wound healing composition in the fourthaspect of Embodiment One (I.D) function together in an unexpectedsynergistic manner to protect and resuscitate mammalian cells.

Accordingly, the combination of ingredients set out in the aboveembodiments functions together in an enhanced manner to prevent andreduce injury to mammalian cells and increase the resuscitation rate ofinjured mammalian cells. The therapeutic effect of the combination ofthe components in each of the above embodiments is markedly greater thanthat expected by the mere addition of the individual therapeuticcomponents. Hence, applicant's therapeutic wound healing compositionsfor treating mammalian cells have the ability to decrease intracellularlevels of hydrogen peroxide production, increase cellular resistance tocytotoxic agents, increase rates of cellular proliferation, and increasecellular viability.

B. Methods For Making The Therapeutic Wound Healing Compositions OfEmbodiment One (I.A-D)

The present invention extends to methods for making the therapeuticwound healing compositions of Embodiment One (I.A-D). In general, atherapeutic wound healing composition is made by forming an admixture ofthe components of the composition. In a first aspect of Embodiment One(I.A), a therapeutic wound healing composition is made by forming anadmixture of (a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells. In a second aspect of Embodiment One (I.B), atherapeutic wound healing composition is made by forming an admixture of(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof,(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof,and (c) a mixture of saturated and unsaturated fatty acids wherein thefatty acids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells. In a third aspect ofEmbodiment One (I.C), a therapeutic wound healing composition is made byforming an admixture of (a) an antioxidant and (b) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells. In a fourth aspect of Embodiment One(I.D), a therapeutic wound healing composition is made by forming anadmixture of (a) lactate selected from the group consisting of lacticacid, pharmaceutically acceptable salts of lactic acid, and mixturesthereof, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes and resuscitation ofmammalian cells.

For some applications, the admixture may be formed in a solvent such aswater, and a surfactant may be added if required. If necessary, the pHof the solvent is adjusted to a range from about 3.5 to about 8.0, andpreferably from about 4.5 to about 7.5, and more preferably about 6.0 toabout 7.4. The admixture is then sterile filtered. Other ingredients mayalso be incorporated into the therapeutic wound healing composition asdictated by the nature of the desired composition as well known by thosehaving ordinary skill in the art. The ultimate therapeutic wound healingcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic wound healing composition (I.A,) for preventingand reducing injury to mammalian cells, and increasing the resuscitationrate of injured mammalian cells, which comprises the steps of admixingthe following ingredients:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells.

C. Methods For Employing The Therapeutic Wound Healing Compositions OfEmbodiment One (I.A-D)

The present invention extends to methods for employing the therapeuticwound healing compositions of Embodiment One (I) in vive and in vitro.In general, a therapeutic wound healing composition is employed bycontacting the therapeutic composition with mammalian cells.

In a first aspect of Embodiment One (I.A), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acid wherein the fatty acid are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a second aspect of Embodiment One (I.B), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured an cells, which comprisesthe steps of (A) providing a therapeutic wound healing composition whichcomprises (a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof, (b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof,and (c) a mixture of saturated and unsaturated fatty acids wherein thefatty acids are those fatty acids required for the resuscitation ofinjured mammalian coils, and (B) contacting the therapeutic woundhealing composition with the mammalian cells.

In a third aspect of Embodiment One (I.C), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) an antioxidant, and (b) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a fourth aspect of Embodiment One (I.D), the invention is directed toa method for preventing and reducing injury to mammalian cells, andincreasing the resuscitation rate of injured mammalian cells, whichcomprises the steps of (A) providing a therapeutic wound healingcomposition which comprises (a) lactate selected from the groupconsisting of lactic acid, pharmaceutically acceptable salts of lacticacid, and mixtures thereof, (b) an antioxidant, and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the resuscitation of injured mammalian cells,and (B) contacting the therapeutic wound healing composition with themammalian cells.

In a preferred embodiment, the invention is directed to a method forhealing a wound in a mammal which comprises the steps of:

(A) providing a therapeutic wound healing composition (I.A) whichcomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacid are those fatty acid required for the resuscitation of injuredmammalian cells; and

(B) contacting the therapeutic wound healing composition with the wound.

The types of wounds which may be healed using the wound healingcompositions of Embodiment One (I.A-D) of the present invention arethose which result from an injury which causes epidermal damage such asincisions, wounds in which the skin is broken by a cutting instrument,and lacerations, wounds in which the skin is broken by a dull or bluntinstrument. The therapeutic compositions may also be used to treatvarious dermatological disorders such as hyperkeratosis, photo-aging,burns, donor site wounds from skin transplants, ulcers (cutaneous,decubitus, venous stasis, and diabetic), psoriasis, skin rashes, andsunburn photoreactive processes. The topical therapeutic compositionsmay also be used orally in the form of a mouth wash or spray to protectand accelerate the healing of injured oral tissue such as mouth soresand burns. The topical therapeutic compositions may further be used inophthalmological preparations to treat wounds such as those which resultfrom corneal ulcers, radialkeratotomy, corneal transplants,epikeratophakia and other surgically induced wounds in the eye. Thetopical therapeutic compositions may in addition be used in anorectalcream and suppositories to treat such conditions as pruritus and,proctitis, anal fissures, and hemorrhoids. In a preferred embodiment,the therapeutic compositions are used to treat wounds such as incisionsand lacerations.

The wound healing compositions of Embodiment One (I.A-D) of the presentinvention may be utilized in topical products, ingestible products, andtissue culture medium to protect mammalian cells and increase theresuscitation rate of injured mammalian cells. For example, thetherapeutic wound healing compositions may be used in topical skin careproducts to protect and increase the resuscitation rate of skin tissuesuch as in the treatment of various dermatological disorders such ashyperkeratosis, photo-aging, and sunburn photoreactive processes. Injuryto skin can occur for a variety of reasons. Injury often occurs toindividuals who wash their hands often, to individuals who are exposedto stressful environmental conditions (overexposure to sun orchemicals), or to the elderly or individuals with an underliningdisease. The addition of the wound healing compositions of the presentinvention to a lotion provides a source of antioxidants to the skinwhich would protect the skin from the harmful effects of UV light,chemicals, and severe drying. The wound healing compositions can be usedfor the following indications: a) Moisturizing and protecting; b)Healing dry cracked skin; c) Treating irritated skin such as diaperrash; d) Healing severe dry skin due to other diseases (venousdermatitis); e) Treating psoriasis and other hyperproliferativediseases; f) Protecting skin from UV light damage (antioxidant skinreplacement); g) Treating seborrheic conditions; and h) Treating shavingwounds in an after shave lotion.

The topical therapeutic wound healing compositions may also be usedorally in the form of a mouth wash or spray to protect and acceleratethe healing of injured oral tissue such as mouth sores and burns. Thetopical therapeutic wound healing compositions may further be used inophthalmological preparations such as eye care products to neutralizehydrogen peroxide used in the cleaning of contact lenses. The topicaltherapeutic wound healing compositions may in addition be used inanorectal creams and suppositories to treat such conditions as pruritusand, proctitis, anal fissures, and hemorrhoids. Initially as white bloodcells enter a wound site, the cells release oxygen radicals, depletingthe antioxidants at the wound site, thus impairing the healing process.Incorporating the wound healing compositions of the present inventioninto a wound healing formulation would facilitate healing by providingthe site with usable antioxidants, and a source of fatty acids neededfor membrane repair. The wound healing compositions can be used for thefollowing indications: a) Healing of cuts and scrapes; b) Burns (healsburns with less scaring and scabbing); c) Decubitus ulcers; d) Bedsores, pressure ulcers; e) Fissures, Hemorrhoids; f) Use in combinationwith immunostimulators (simulated healing in healing deficient people);g) Post surgical wounds; h) Bandages; i) Diabetic ulcers; j) Venousulceration; and k) Use in combination with wound cleansing agents.

The therapeutic wound healing compositions may also be used iningestible products to protect and increase the resuscitation rate oferosions, stomach ulcers, and hemorrhages in the gastric mucosa. Otheringestible therapeutic products include: stroke medications; autoimmunedisease medications; arthritis medications; ulcer medications; cancermedications (cytotoxic agents); heart medication to improve regionalventricular function and restore normal heart rate and pressurefunctions; lung medication to repair injured tissue; liver medication tosuppress lipogenesis of alcoholic origin and prevent hepatic steatosis;kidney medication to suppress urinary calculi (kidney stones);detoxification medication to antagonize heavy metal poisoning, cyanidepoisoning, sodium sulfide poisoning, other types of poisoning,; andreduce and neutralize the production of oxygen radicals which producesinjury to tissue, to protect and further enhance the resuscitation rateof the injured mammalian cells. The therapeutic wound healingcompositions may be used in ingestible products to treat inflammatorydiseases such as hepatitis, gastritis, colitis, esophagitis, arthritis,and pancreatitis.

The therapeutic wound healing compositions of the present invention mayalso be used in tissue culture media and organ transplant media toprevent and reduce injury to mammalian cells and increase theresuscitation rate of injured mammalian cells. Tissue cultures andtransplant organs encounter reactive oxygen species generated in theculture media by the injured cells. Organs particularly susceptible tooxidative damage during transport and transplantation due to reperfusioninjury following ischemia are corneas, livers, hearts, and kidneys. Thetherapeutic wound healing compositions may be useful to abrogatereperfusion injury to such transplant organs.

In a specific embodiment, the invention is directed to a method forpreserving mammalian cells in a culture medium which comprises the stepsof:

(A) providing a therapeutic wound healing composition selected from thegroup of consisting of:

(I.A) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty adds wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acid required for the repair of cellular membranesand resuscitation of mammalian cells;

(I.C) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D) (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the resuscitation of injuredmammalian cells;

(B) providing mammalian cells in a culture medium; and

(C) contacting the therapeutic wound healing composition from step (A)with the mammalian cells in the culture medium from step (B).

D. Fomulations Of The Therapeutic Wound Healing Compositions OfEmbodiment One (I.A-D)

Once prepared, the inventive therapeutic wound healing compositions ofEmbodiment One (I.A-D) may be stored for future use or may be formulatedin effective amounts with pharmaceutically acceptable carriers toprepare a wide variety of pharmaceutical compositions. Examples ofpharmaceutically acceptable carriers are pharmaceutical appliances,topical vehicles (non-oral and oral), and ingestible vehicles.

Examples of pharmaceutical appliances are sutures, staples, gauze,bandages, burn dressings, artificial skins, liposome or micellformulations, microcapsules, aqueous vehicles for soaking gauzedressings, and the like, and mixtures thereof. Non-oral topicalcompositions employ non-oral topical vehicles, such as creams, gelsformulations, foams, ointments and sprays, salves, and films, which areintended to be applied to the skin or body cavity and are not intendedto be taken by mouth. Oral topical compositions employ oral vehicles,such as mouthwashes, rinses, oral sprays, suspensions, and dental gels,which are intended to be taken by mouth but are not intended to beingested. Ingestible compositions employ ingestible or partly ingestiblevehicles such as confectionery bulking agents which include hard andsoft confectionery such as lozenges, tablets, toffees, nougats,suspensions, chewy candies, and chewing gums.

In one form of the invention, the therapeutic wound healing compositionis incorporated into a pharmaceutical appliance which may be in the formof sutures, staples, gauze, bandages, burn dressings, artificial skins,liposome or micell formulations, microcapsules, aqueous vehicles forsoaking gauze dressings, and the like, and mixtures thereof. A varietyof traditional ingredients may optionally be included in thepharmaceutical composition in effective amounts such as buffers,preservatives, tonicity adjusting agents, antioxidants, polymers foradjusting viscosity or for use as extenders, and excipients, and thelike. Specific illustrative examples of such traditional ingredientsinclude acetate and borate buffers; thimerosal, sorbic acid, methyl andpropyl paraben and chlorobutanol preservatives; sodium chloride andsugars to adjust the tonicity; and excipients such as mannitol, lactoseand sucrose. Other conventional pharmaceutical additives known to thosehaving ordinary skill in the pharmaceutical arts may also be used in thepharmaceutical composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe employed in the pharmaceutical appliance. These amounts are readilydetermined by those skilled in the art without the need for undueexperimentation. The exact amount of the therapeutic wound healingcomposition employed is subject to such factors as the type andconcentration of the therapeutic wound healing composition and the typeof pharmaceutical appliance employed. Thus, the mount of therapeuticwound healing composition may be varied in order to obtain the resultdesired in the final product and such variations are within thecapabilities of those skilled in the art without the need for undueexperimentation. In a preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, by weight of the pharmaceuticalcomposition. In a more preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 3%, by weight of the pharmaceuticalcomposition. In a most preferred embodiment, the pharmaceuticalcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 1%, by weight of the pharmaceuticalcomposition.

The present invention extend to methods for making the pharmaceuticalcompositions. In general, a pharmaceutical composition is made bycontacting a therapeutically effective amount of a therapeutic woundhealing composition with a pharmaceutical appliance and the otheringredients of the final desired pharmaceutical composition. Thetherapeutic wound healing composition may be in a solvent and may beabsorbed onto a pharmaceutical appliance.

Other ingredients will usually be incorporated into the composition asdictated by the nature of the desired composition as well known by thosehaving ordinary skill in the art. The ultimate pharmaceuticalcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In another form of the invention, the therapeutic wound healingcomposition is incorporated into a non-oral topical vehicle which may bein the form of a cream, gel, foam, ointment, spray, and the like.Typical non-toxic non-oral topical vehicles known in the pharmaceuticalarts may be used in the present invention. The preferred non-oraltopical vehicles are water and pharmaceutically acceptablewater-miscible organic solvents such as ethyl alcohol, isopropylalcohol, propylene glycol, glycerin, and the like, and mixtures of thesesolvents. Water-alcohol mixtures are particularly preferred and aregenerally employed in a weight ratio from about 1:1 to about 20:1,preferably from about 3:1 to about 20:1, and most preferably from about3:1 to about 10:1, respectively.

The non-oral topical therapeutic wound healing compositions may alsocontain conventional additives employed in those products. Conventionaladditives include humectants, emollients, lubricants, stabilizers, dyes,and perfumes, providing the additives do not interfere with thetherapeutic properties of the therapeutic wound healing composition.

Suitable humectants useful in the non-oral topical therapeutic woundhealing compositions include glycerin, propylene glycol, polyethyleneglycol, sorbitan, fructose, and the like, and mixtures thereof.Humectants, when employed, may be present in amounts from about 10% toabout 20%, by weight of the topical therapeutic wound healingcomposition.

The coloring agents (colors, colorants) useful in the non-oral topicaltherapeutic wound healing composition are used in amounts effective toproduce the desired color. These coloring agents include pigments whichmay be incorporated in amounts up to about 6% by weight of the non-oraltopical therapeutic wound healing composition. A preferred pigment,titanium dioxide, may be incorporated in amounts up to about 2%, andpreferably less than about 1%, by weight of the non-oral topicaltherapeutic wound healing composition. The coloring agents may alsoinclude natural food colors and dyes suitable for food, drug andcosmetic applications. These coloring agents are known as F.D.& C. dyesand lakes. The materials acceptable for the foregoing uses arepreferably water-soluble. Illustrative nonlimiting examples include theindigoid dye known as F.D.& C. Blue No.2, which is the disodium salt of5,5-indigotindisulfonic acid. Similarly, the dye known as F.D.& C. GreenNo. 1 comprises a triphenylmethane dye and is the monosodium salt of 4-4-(N-ethyl-p-sulfoniumbenzylamino) diphenylmethylene!-1-(N-ethyl-N-p-sulfoniumbenzyl)-delta-2,5-cyclohexadieneimine!. A fullrecitation of all F.D.& C. coloring agents and their correspondingchemical structures may be found in the Kirk-Othmer Encyclopedia ofChemical Technology, 3rd Edition, in volume 5 at pages 857-884, whichtext is incorporated herein by reference.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed with a non-oral topical vehicle to form a topical therapeuticwound healing composition. These amounts are readily determined by thoseskilled in the art without the need for undue experimentation. In apreferred embodiment, the non-oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 10% and a non-oral topical vehicle ina quantity sufficient to bring the total amount of composition to 100%,by weight of the non-oral topical therapeutic wound healing composition.In a more preferred embodiment, the non-oral topical therapeutic woundhealing compositions will comprise the therapeutic wound healingcomposition in an amount from about 0.1% to about 5%, and in a mostpreferred embodiment, the non-oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 2%, and a non-oral topical vehicle ina quantity sufficient to bring the total amount of composition to 100%,by weight of the non-oral topical therapeutic wound healing composition.

The present invention extends to methods for preparing the non-oraltopical therapeutic wound healing compositions. In such a method, thenon-oral topical therapeutic wound healing composition is prepared byadmixing a therapeutically effective amount of the therapeutic woundhealing composition of the present invention and a non-oral topicalvehicle. The final compositions are readily prepared using standardmethods and apparatus generally known by those skilled in thepharmaceutical arts. The apparatus useful in accordance with the presentinvention comprises mixing apparatus well known in the pharmaceuticalarts, and therefore the selection of the specific apparatus will beapparent to the artisan.

In another form of the invention, the therapeutic wound healingcomposition is incorporated into an oral topical vehicle which may be inthe form of a mouthwash, rinse, oral spray, suspension, dental gel, andthe like. Typical non-toxic oral vehicles known in the pharmaceuticalarts may be used in the present invention. The preferred oral vehiclesare water, ethanol, and water-ethanol mixtures. The water-ethanolmixtures are generally employed in a weight ratio from about 1:1 toabout 20:1, preferably from about 3:1 to about 20:1, and most preferablyfrom about 3:1 to about 10:1, respectively. The pH value of the oralvehicle is generally from about 4 to about 7, and preferably from about5 to about 6.5. An oral topical vehicle having a pH value below about 4is generally irritating to the oral cavity and an oral vehicle having apH value greater than about 7 generally results in an unpleasant mouthfeel.

The oral topical therapeutic wound healing compositions may also containconventional additives normally employed in those products. Conventionaladditives include a fluorine providing compound, a sweetening agent, aflavoring agent, a coloring agent, a humectant, a buffer, and anemulsifier, providing the additives do not interfere with thetherapeutic properties of the therapeutic wound healing composition.

The coloring agents and humectants, and the amounts of these additivesto be employed, set out above as useful in the non-oral topicaltherapeutic wound healing composition may be used in the oral topicaltherapeutic wound healing composition.

Fluorine providing compounds may be fully or slightly water soluble andare characterized by their ability to release fluoride ions or fluoridecontaining ions in water and by their lack of reaction with othercomponents in the composition. Typical fluorine providing compounds areinorganic fluoride salts such as water-soluble alkali metal, alkalineearth metal, and heavy metal salts, for example, sodium fluoride,potassium fluoride, ammonium fluoride, cuprous fluoride, zinc fluoride,stannic fluoride, stannous fluoride, barium fluoride, sodiumfluorosilicate, ammonium fluorosilicate, sodium fluorozirconate, sodiummonofluorophosphate, aluminum mono- and di-fluorophosphates andfluorinated sodium calcium pyrophosphate. Alkali metal fluorides, tinfluoride and monofluorophosphates, such as sodium and stannous fluoride,sodium monofluorophosphate and mixtures thereof, are preferred.

The amount of fluorine providing compound present in the present oraltopical therapeutic wound healing composition is dependent upon the typeof fluorine providing compound employed, the solubility of the fluorinecompound, and the nature of the final oral therapeutic wound healingcomposition. The amount of fluorine providing compound used must be anontoxic amount. In general, the fluorine providing compound when usedwill be present in an amount up to about 1%, preferably from about0.001% to about 0.1%, and most preferably from about 0.001% to about0.05%, by weight of the oral topical therapeutic wound healingcomposition.

When sweetening agents (sweeteners) are used, those sweeteners wellknown in the art, including both natural and artificial sweeteners, maybe employed. The sweetening agent used may be selected from a wide rangeof materials including water-soluble sweetening agents, water-solubleartificial sweetening agents, water-soluble sweetening agents derivedfrom naturally occurring water-soluble sweetening agents, dipeptidebased sweetening agents, and protein based sweetening agents, includingmixtures thereof. Without being limited to particular sweetening agents,representative categories and examples include:

(a) water-soluble sweetening agents such as monosaccharides,disaccharides and polysaccharides such as xylose, ribose,glucose(dextrose), mannose, galactose, fructose (levulose), sucrose(sugar), maltose, invert sugar (a mixture of fructose and glucosederived from sucrose), partially hydrolyzed starch, corn syrup solids,dihydrochalcones, monellin, steviosides, and glycyrrhizin, and mixturesthereof;

(b) water-soluble artificial sweeteners such as soluble saccharin salts,i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium,ammonium or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassiumsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like;

(c) dipeptide based sweeteners, such as L-aspartic acid derivedsweeteners, such as L-aspartyl-L-phenylalanine methyl ester (Aspaname)and materials described in U.S. Pat. No. 3,492,131,L-Alpha-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alanin-amidehydrate (Alitame), methyl esters of L-aspartyl-L-phenylglycerine andL-aspartyl-L-2,5-dihydrophenyl-glycine,L-aspartyl-2,5-dihydro-L-phenylalanine;L-aspartyl-L-(1-cyclohexen)-alanine, and the like;

(d) water-soluble sweeteners derived from naturally occurringwater-soluble sweeteners, such as chlorinated derivatives of ordinarysugar (sucrose), e.g., chlorodeoxysugar derivatives such as derivativesof chlorodeoxysucrose or chlorodeoxygalactosucrose, known, for example,under the product designation of Sucralose; examples ofchlorodeoxysucrose and chlorodeoxygalacto-sucrose derivatives includebut are not limited to: 1-chloro-1'-deoxysucrose;4-chloro-4-deoxy-Alpha-D-galacto-pyranosyl-Alpha-D -fructofuranoside, or4-chloro-4-deoxygalactosucrose;4-chloro-4-deoxy-Alpha-D-galacto-pyranosyl-1-chloro-1-deoxy-β-D-fructo-furanoside, or4,1'-dichloro-4,1'-dideoxygalactosucrose;1,6'-dichloro-1,6'-dideoxysucrose;4-chloro-4-deoxy-Alpha-D-galacto-pyranosyl-1,6-dichloro-1,6-dideoxy-β-D-fructo-furanoside,or 4,1',6'-trichloro-4,1',6'-trideoxygalacto-sucrose;4,6-dichloro-4,6-dideoxy-Alpha-D-galacto-pyranosyl-6-chloro-6-deoxy-β-D-fructofuranoside,or 4,6,6'-trichloro-4,6,6'-trideoxygalactosucrose;6,1',6'-trichloro-6,1',6'-trideoxysucrose;4,6-dichloro-4,6-dideoxy-Alpha-D-galacto-pyranosyl-1,6-dichloro-1,6-di-deoxy-β-D-fructofuranoside,or 4,6,1',6'-tetrachloro-4,6,1',6'-tetradeoxygalacto-sucrose; and4,6,1',6'-tetrachloro-4,6,1',6'-tetradeoxy-sucrose; and

(e) protein based sweeteners such as thaumaoccous danielli (Thaumatin Iand II).

In general, an effective amount of sweetening agent is utilized toprovide the level of sweetness desired in the particular oral topicaltherapeutic wound healing composition, and this amount will vary withthe sweetener selected and the final oral therapeutic product desired.The amount of sweetener normally present is in the range from about0.0025% to about 90%, by weight of the oral topical therapeutic woundhealing composition, depending upon the sweetener used. The exact rangeof amounts for each type of sweetener is well known in the art and isnot the subject of the present invention.

The flavoring agents (flavors, flavorants) which may be used includethose flavors known to the skilled artisan, such as natural andartificial flavors. Suitable flavoring agents include mints, such aspeppermint, citrus flavors such as orange and lemon, artificial vanilla,cinnamon, various fruit flavors, both individual and mixed, and thelike.

The amount of flavoring agent employed in the oral topical therapeuticwound healing composition is normally a matter of preference subject tosuch factors as the type of final oral therapeutic wound healingcomposition, the individual flavor employed, and the strength of flavordesired. Thus, the amount of flavoring may be varied in order to obtainthe result desired in the final product and such variations are withinthe capabilities of those skilled in the art without the need for undueexperimentation. The flavoring agents, when used, are generally utilizedin amounts that may, for example, range in amounts from about 0.05% toabout 6%, by weight of the oral topical therapeutic wound healingcomposition.

Suitable buffer solutions useful in the non-oral topical therapeuticwound healing compositions include citric acid-sodium citrate solution,phosphoric acid-sodium phosphate solution, and acetic acid-sodiumacetate solution in amounts up to about 1%, and preferably from about0.05% to about 0.5% by weight of the oral topical therapeutic woundhealing composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed with an oral topical vehicle to form a topical therapeuticwound healing composition. These amounts are readily determined by thoseskilled in the art without the need for undue experimentation. In apreferred embodiment, the oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 10% and a oral topical vehicle in aquantity sufficient to bring the total amount of composition to 100%, byweight of the oral topical therapeutic wound healing composition. In amore preferred embodiment, the oral topical therapeutic wound healingcompositions will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, and in a most preferredembodiment, the oral topical therapeutic wound healing compositions willcomprise the therapeutic wound healing composition in an amount fromabout 0.1% to about 2%, and a oral topical vehicle in a quantitysufficient to bring the total amount of composition to 100%, by weightof the oral topical therapeutic wound healing composition.

The present invention extends to methods for preparing the oral topicaltherapeutic wound healing compositions. In such a method, the oraltopical therapeutic wound healing composition is prepared by admixing atherapeutically effective amount of the therapeutic wound healingcomposition of the present invention and an oral topical vehicle. Thefinal compositions are readily prepared using standard methods andapparatus generally known by those skilled in the pharmaceutical arts.The apparatus useful in accordance with the present invention comprisesmixing apparatus well known in the pharmaceutical arts, and thereforethe selection of the specific apparatus will be apparent to the artisan.

In a preferred embodiment, an oral topical therapeutic wound healingcomposition is made by first dissolving coloring agents, sweeteningagents, and similar additives in water. The therapeutic wound healingcomposition is then admixed with the aqueous solution. Then sufficientwater or ethanol, or mixtures of water and ethanol, are added to thesolution with mixing until the final solution volume is reached. In amore preferred embodiment, the therapeutic wound healing composition isadded to the solution as the final ingredient. The final oval topicaltherapeutic wound healing compositions are readily prepared usingmethods generally known in the pharmaceutical arts.

The oral therapeutic wound healing composition may also be in the formof dental gel. As used herein, the term "gel" means a solid or semisolidcolloid which contains considerable quantities of water. The colloidparticles in a gel are linked together in a coherent meshwork whichimmobilizes the water contained inside the meshwork.

The dental gel compositions of the present invention may contain theconventional additives set out above for oral topical therapeutic woundhealing compositions such as mouthwashes, rinses, oral sprays, andsuspensions and, in addition, may contain additional additives such as apolishing agent, a desensitizing agent, and the like, providing theadditional additives do not interfere with the therapeutic properties ofthe therapeutic wound healing composition.

In a dental gel composition, the oral vehicle generally comprises water,typically in an amount from about 10% to about 90%, by weight of thedental gel composition. Polyethylene glycol, propylene glycol, glycerin,and mixtures thereof may also be present in the vehicle as humectants orbinders in amounts from about 18% to about 30%, by weight of the dentalgel composition. Particularly preferred oral vehicles comprise mixturesof water with polyethylene glycol or water with glycerin andpolypropylene glycol.

The dental gels of the present invention include a gelling agent(thickening agent) such as a natural or synthetic gum or gelatin.Gelling agents such as hydroxyethyl cellulose, methyl cellulose,glycerin, carboxypolymethylene, and gelatin and the like, and mixturesthereof may be used. The preferred gelling agent is hydroxyethylcellulose. Gelling agents may be used in amounts from about 0.5% toabout 5%, and preferably from about 0.5% to about 2%, by weight of thedental gel composition.

The dental gel compositions of the present invention may also include apolishing agent. In clear gels, a polishing agent of colloidal silicaand/or alkali metal aluminosilicate complexes is preferred since thesematerials have refractive indices close to the refractive indices of thegelling systems commonly used in dental gels. In non-clear gels, apolishing agent of calcium carbonate or calcium dihydrate may be used.These polishing agents may be used in amounts up to about 75%, andpreferably in amounts up to about 50%, by weight of the dental gelcomposition.

The dental gel may also contain a desensitizing agent such as acombination of citric acid and sodium citrate. Citric acid may be usedin an amount from about 0.1% to about 3%, and preferably from about 0.2%to about 1%, by weight, and sodium citrate may be used in an amount fromabout 0.3% to about 9%, and preferably from about 0.6% to about 3%, byweight of the dental gel composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into the dental gel compositions. These amounts are readilydetermined by those skilled in the art without the need for undueexperimentation. In a preferred embodiment, the dental gel compositionswill comprise the therapeutic wound healing composition in an amountfrom about 0.1% to about 10% and an oral topical vehicle in a quantitysufficient to bring the total amount of composition to 100%, by weightof the dental gel composition. In a more preferred embodiment, thedental gel compositions will comprise the therapeutic wound healingcomposition in an amount from about 0.1% to about 5%, and in a mostpreferred embodiment, the dental gel compositions will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 2%, and an oral topical vehicle in a quantity sufficient to bringthe total amount of composition to 100%, by weight of the dental gelcomposition.

The present invention extends to methods for preparing the therapeuticdental gel compositions. In such a method, the dental gel composition isprepared by admixing a therapeutically effective amount of thetherapeutic wound healing composition of the present invention and anoral topical vehicle. The final compositions are readily prepared usingmethods generally known by those skilled in the dental andpharmaceutical arts. The apparatus useful in accordance with the presentinvention comprises mixing apparatus well known in the pharmaceuticalarts, and therefore the selection of the specific apparatus will beapparent to the artisan.

In a preferred embodiment, a therapeutic dental gel composition is madeby first dispersing a gelling agent in a humectant or water, or amixture of both, then admixing to the dispersion an aqueous solution ofthe water-soluble additives such as the fluorine providing compound,sweeteners and the like, then adding the polishing agent, and lastlyadmixing the flavoring agent and the therapeutic wound healingcomposition. The final gel mixture is then tubed or otherwise packaged.The liquids and solids in a gel product are proportioned to form acreamy or gelled mass which is extrudable from a pressurized containeror from a collapsible tube. The final therapeutic wound healingcompositions are readily prepared using methods generally known in thepharmaceutical arts.

In yet another form of the invention, the therapeutic wound healingcomposition is incorporated into an ingestible vehicle. The ingestiblevehicle may be a confectionery bulking agent in the form of lozenges,tablets, toffees, nougats, suspensions, chewy candies, chewing gums, andthe like. The pharmaceutically acceptable carriers may be prepared froma wide range of materials including, but not limited to, diluents,binders and adhesives, lubricants, disintegrants, coloring agents,bulking agents, flavoring agents, sweetening agents and miscellaneousmaterials such as buffers and adsorbents that may be needed in order toprepare a particular therapeutic confection.

The preparation of confectionery formulations is historically well knownand has changed little through the years. Confectionery items have beenclassified as either "hard" confectionery or "soft" confectionery. Thetherapeutic wound healing compositions of the present invention can beincorporated into confectionery compositions by admixing the inventivecomposition into conventional hard and soft confections.

As used herein, the term confectionery material means a productcontaining a bulking agent selected from a wide variety of materialssuch as sugar, corn syrup, and in the case of sugarless bulking agents,sugar alcohols such as sorbitol and mannitol and mixtures thereof.Confectionery material may include such exemplary substances aslozenges, tablets, toffee, nougat, suspensions, chewy candy, chewing gumand the like. The bulking agent is present in a quantity sufficient tobring the total amount of composition to 100%. In general, the bulkingagent will be present in amounts up to about 99.98%, preferably inamounts up to about 99.9%, and more preferably in amounts up to about99%, by weight of the ingestible therapeutic wound healing composition.

Lozenges are flavored medicated dosage forms intended to be sucked andheld in the mouth. Lozenges may be in the form of various shapes such asflat, circular, octagonal and biconvex forms. The lozenge bases aregenerally in two forms: hard boiled candy lozenges and compressed tabletlozenges.

Hard boiled candy lozenges may be processed and formulated byconventional means. In general, a hard boiled candy lozenge has a basecomposed of a mixture of sugar and other carbohydrate bulking agentskept in an amorphous or glassy condition. This amorphous or glassy formis considered a solid syrup of sugars generally having from about 0.5%to about 1.5% moisture. Such materials normally contain up to about 92%corn syrup, up to about 55% sugar and from about 0.1% to about 5% water,by weight of the final composition. The syrup component is generallyprepared from corn syrups high in fructose, but may include othermaterials. Further ingredients such as flavoring agents, sweeteningagents, acidulants, coloring agents and the like may also be added.

Boiled candy lozenges may also be prepared from non-fermentable sugarssuch as sorbitol, mannitol, and hydrogenated corn syrup. Typicalhydrogenated corn syrups are Lycasin, a commercially available productmanufactured by Roquette Corporation, and Hystar, a commerciallyavailable product manufactured by Lonza, Inc. The candy lozenges maycontain up to about 95% sorbitol, a mixture of sorbitol and mannitol ina ratio from about 9.5:0.5 up to about 7.5:2.5, and hydrogenated cornsyrup up to about 55%, by weight of the solid syrup component.

Boiled candy lozenges may be routinely prepared by conventional methodssuch as those involving fire cookers, vacuum cookers, andscraped-surface cookers also referred to as high speed atmosphericcookers.

Fire cookers involve the traditional method of making a boiled candylozenge base. In this method, the desired quantity of carbohydratebulking agent is dissolved in water by heating the agent in a kettleuntil the bulking agent dissolves. Additional bulking agent may then beadded and cooking continued until a final temperature of 145° C. to 156°C. is achieved. The batch is then cooled and worked as a plastic-likemass to incorporate additives such as flavors, colorants and the like.

A high-speed atmospheric cooker uses a heat-exchanger surface whichinvolves spreading a film of candy on a heat exchange surface, the candyis heated to 165° C. to 170° C. in a few minutes. The candy is thenrapidly cooled to 100° C. to 120° C. and worked as a plastic-like massenabling incorporation of the additives, such as flavors, colorants andthe like.

In vacuum cookers, the carbohydrate bulking agent is boiled to 125° C.to 132° C., vacuum is applied and additional water is boiled off withoutextra heating. When cooking is complete, the mass is a semi-solid andhas a plastic-like consistency. At this point, flavors, colorants, andother additives are admixed in the mass by routine mechanical mixingoperations.

The optimum mixing required to uniformly mix the flavoring agents,coloring agents and other additives during conventional manufacturing ofboiled candy lozenges is determined by the time needed to obtain auniform distribution of the materials. Normally, mixing times of from 4to 10 minutes have been found to be acceptable.

Once the boiled candy lozenge has been properly tempered it may be cutinto workable portions or formed into desired shapes. A variety offorming techniques may be utilized depending upon the shape and size ofthe final product desired. A general discussion of the composition andpreparation of hard confections may be found in H. A. Lieberman,Pharmaceutical Dosage Forms: Tablets, Volume I (1980), Marcel Dekker,Inc., New York, N.Y. at pages 339 to 469, which disclosure isincorporated herein by reference.

The apparatus useful in accordance with the present invention comprisescooking and mixing apparatus well known in the confectionerymanufacturing arts, and therefore the selection of the specificapparatus will be apparent to the artisan.

In contrast, compressed tablet confections contain particulate materialsand are formed into structures under pressure. These confectionsgenerally contain sugars in amounts up to about 95%, by weight of thecomposition, and typical tablet excipients such as binders andlubricants as well as flavoring agents, coloring agents and the like.

In addition to hard confectionery materials, the lozenges of the presentinvention may be made of soft confectionery materials such as thosecontained in nougat. The preparation of soft confections, such asnougat, involves conventional methods, such as the combination of twoprimary components, namely (1) a high boiling syrup such as a cornsyrup, hydrogenated starch hydrolysate or the like, and (2) a relativelylight textured frappe, generally prepared from egg albumin, gelatin,vegetable proteins, such as soy derived compounds, sugarless milkderived compound such as milk proteins, and mixtures thereof. The frappeis generally relatively light, and may, for example, range in densityfrom about 0.5 to about 0.7 grams/cc.

The high boiling syrup, or "bob syrup" of the soft confectionery isrelatively viscous and has a higher density than the frappe component,and frequently contains a substantial amount of carbohydrate bulkingagent such as a hydrogenated starch hydrolysate. Conventionally, thefinal nougat composition is prepared by the addition of the "bob syrup"to the frappe under agitation, to form the basic nougat mixture. Furtheringredients such as flavoring agents, additional carbohydrate bulkingagent, coloring agents, preservatives, medicaments, mixtures thereof andthe like may be added thereafter also under agitation. A generaldiscussion of the composition and preparation of nougat confections maybe found in B. W. Minifie, Chocolate: Cocoa and Confectionery: Scienceand Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn.(1980), at pages 424-425, which disclosure is incorporated herein byreference.

The procedure for preparing the soft confectionery involves knownprocedures. In general, the frappe component is prepared first andthereafter the syrup component is slowly added under agitation at atemperature of at least about 65° C., and preferably at least about 100°C. The mixture of components is continued to be mixed to form a uniformmixture, after which the mixture is cooled to a temperature below 80°C., at which point, the flavoring agent may be added. The mixture isfurther mixed for an additional period until it is ready to be removedand formed into suitable confectionery shapes.

The ingestible therapeutic wound healing compositions may also be in theform of a pharmaceutical suspension. Pharmaceutical suspensions of thisinvention may be prepared by conventional methods long established inthe art of pharmaceutical compounding. Suspensions may contain adjunctmaterials employed in formulating the suspensions of the art. Thesuspensions of the present invention can comprise:

(a) preservatives such as butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), benzoic acid, ascorbic acid, methyl paraben,propyl paraben, tocopherols, and the like, and mixtures thereof.Preservatives are generally present in amounts up to about 1%, andpreferably from about 0.05% to about 0.5%, by weight of the suspension;

(b) buffers such as citric acid-sodium citrate, phosphoric acid-sodiumphosphate, and acetic acid-sodium acetate in amounts up to about 1%, andpreferably from about 0.05% to about 0.5%, by weight of the suspension;

(c) suspending agents or thickeners such as cellulosics likemethylcellulose, carrageenans like alginic acid and its derivatives,xanthan gums, gelatin, acacias, and microcrystalline cellulose inamounts up to about 20%, and preferably from about 1% to about 15%, byweight of the suspension;

(d) antifoaming agents such as dimethyl polysiloxane in amounts up toabout 0.2%, and preferably from about 0.01% to about 0.1%, by weight ofthe suspension;

(e) sweetening agents such as those sweeteners well known in the art,including both natural and artificial sweeteners. Sweetening agents suchas monosaccharides, disaccharides and polysaccharides such as xylose,ribose, glucose (dextrose), mannose, galactose, fructose (levulose),sucrose (sugar), maltose, invert sugar (a mixture of fructose andglucose derived from sucrose), partially hydrolyzed starch, corn syrupsolids, dihydrochalcones, monellin, steviosides, glycyrrhizin and sugaralcohols such as sorbitol, mannitol, maltitol, hydrogenated starchhydrolysates and mixtures thereof may be utilized in amounts up to about60%, and preferably from about 20% to about 50%, by weight of thesuspension. Water-soluble artificial sweeteners such as solublesaccharin salts, i.e., sodium or calcium saccharin salts, cyclamatesalts, the sodium, ammonium or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassiumsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like may beutilized in amounts from about 0.001% to about 5%, by weight of thesuspension;

(f) flavoring agents such as those flavors well known to the skilledartisan, such as natural and artificial flavors and mints, such aspeppermint, menthol, citrus flavors such as orange and lemon, artificialvanilla, cinnamon, various fruit flavors, both individual and mixed andthe like may be utilized in amounts from about 0.5% to about 5%, byweight of the suspension;

(g) coloring agents such as pigments which may be incorporated inamounts up to about 6%, by weight of the suspension. A preferredpigment, titanium dioxide, may be incorporated in amounts up to about2%, and preferably less than about 1%, by weight of the suspension. Thecoloring agents may also include natural food colors and dyes suitablefor food, drug and cosmetic applications. These colorants are known asF.D.& C. dyes and lakes. The materials acceptable for the foregoing usesare preferably water-soluble. Such dyes are generally present in amountsup to about 0.25%, and preferably from about 0.05% to about 0.2%, byweight of the suspension;

(h) decolorizing agents such as sodium metabisulfite, ascorbic acid andthe like may be incorporated into the suspension to prevent colorchanges due to aging. In general, decolorizing agents may be used inamounts up to about 0.25%, and preferably from about 0.05% to about0.2%, by weight of the suspension; and

(i) solubilizers such as alcohol, propylene glycol, polyethylene glycol,and the like may be used to solubilize the flavoring agents. In general,solubilizing agents may be used in amounts up to about 10%, andpreferably from about 2% to about 5%, by weight of the suspension.

The pharmaceutical suspensions of the present invention may be preparedas follows:

(A) admix the thickener with water heated from about 40° C. to about 95°C., preferably from about 40° C. to about 70° C., to form a dispersionif the thickener is not water soluble or a solution if the thickener iswater soluble;

(B) admix the sweetening agent with water to form a solution;

(C) admix the therapeutic wound healing composition with thethickener-water admixture to form a uniform thickener-therapeutic woundhealing composition;

(D) combine the sweetener solution with the thickener-therapeutic woundhealing composition and mix until uniform; and

(E) admix the optional adjunct materials such as coloring agents,flavoring agents, decolorants, solubilizers, antifoaming agents, buffersand additional water with the mixture of step (D) to form thesuspension.

The ingestible therapeutic wound healing compositions of this inventionmay also be in chewable form. To achieve acceptable stability andquality as well as good taste and mouth feel in a chewable formulationseveral considerations are important. These considerations include theamount of active substance per tablet, the flavoring agent employed, thedegree of compressibility of the tablet and the organoleptic propertiesof the composition.

Chewable therapeutic candy is prepared by procedures similar to thoseused to make soft confectionery. In a typical procedure, a boiledsugar-corn syrup blend is formed to which is added a frappe mixture. Theboiled sugar-corn syrup blend may be prepared from sugar and corn syrupblended in parts by weight ratio of about 90:10 to about 10:90. Thesugar-corn syrup blend is heated to temperatures above about 120° C. toremove water and to form a molten mass. The frappe is generally preparedfrom gelatin, egg albumin, milk proteins such as casein, and vegetableproteins such as soy protein, and the like, which is added to a gelatinsolution and rapidly mixed at ambient temperature to form an aeratedsponge like mass. The frappe is then added to the molten candy mass andmixed until homogeneous at temperatures between about 65° C. and about120° C.

The ingestible therapeutic wound healing composition of the instantinvention can then be added to the homogeneous mixture as thetemperature is lowered to about 65° C.-95° C. whereupon additionalingredients can then be added such as flavoring agents and coloringagents. The formulation is further cooled and formed into pieces ofdesired dimensions.

A general discussion of the lozenge and chewable tablet forms ofconfectionery may be found in H. A. Lieberman and L. Lachman,Pharmaceutical Dosage Forms: Tablets Volume 1, Marcel Dekker, Inc., NewYork, N.Y. at pages 289 to 466, which disclosure is incorporated hereinby reference.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into the hard and soft confectionery products. These amountsare readily determined by those skilled in the art without the need forundue experimentation. In a preferred embodiment, the ingestibletherapeutic wound healing composition will comprise the therapeuticwound healing composition in an amount from about 0.1% to about 10% andan ingestible vehicle, that is a pharmaceutically acceptable carrier, ina quantity sufficient to bring the total amount of composition to 100%,by weight the ingestible therapeutic wound healing composition. In amore preferred embodiment, the ingestible composition will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 5%, and in a most preferred embodiment, the ingestible compositionwill comprise the therapeutic wound healing composition in an amountfrom about 0.1% to about 2%, and an ingestible vehicle in a quantitysufficient to bring the total amount of composition to 100%, by weightthe ingestible therapeutic wound healing composition.

The present invention extends to methods of making the ingestibletherapeutic wound healing compositions. In such methods, an ingestibletherapeutic wound healing composition is prepared by admixing atherapeutically effective amount of the therapeutic wound healingcomposition with a pharmaceutically-acceptable carrier. The apparatususeful in accordance with the present invention comprises mixing andheating apparatus well known in the confectionery arts, and thereforethe selection of the specific apparatus will be apparent to the artisan.The final ingestible therapeutic wound healing compositions are readilyprepared using methods generally known in the confectionery arts.

The therapeutic wound healing compositions may also be incorporated intochewing gums. In this form of the invention, the chewing gum compositioncontains a gum base, a bulking agent, the inventive therapeutic woundhealing composition, and various additives.

The gum base employed will vary greatly depending upon various factorssuch as the type of base desired, the consistency of gum desired and theother components used in the composition to make the final chewing gumproduct. The gum base may be any water-insoluble gum base known in theart, and includes those gum bases utilized for chewing gums and bubblegums. Illustrative examples of suitable polymers in gum bases includeboth natural and synthetic elastomers and rubbers. For example, thosepolymers which are suitable as gum bases include, without limitation,substances of vegetable origin such as chicle, crown gum, nispero,rosadinha, jelutong, perillo, niger gutta, tunu, balata, gutta-percha,lechi-capsi, sorva, gutta kay, mixtures thereof and the like. Syntheticelastomers such as butadien styrene copolymers, polyisobutylene,isobutylene-isoprene copolymers, polyethylene, mixtures thereof and thelike are particularly useful.

The gum base may include a non-toxic vinyl polymer, such as polyvinylacetate and its partial hydrolysate, polyvinyl alcohol, and mixturesthereof. When utilized, the molecular weight of the vinyl polymer mayrange from about 2,000 up to and including about 94,000.

The amount of gum base employed will vary greatly depending upon variousfactors such as the type of base used, the consistency of the gumdesired and the other components used in the composition to make thefinal chewing gum product. In general, the gum base will be present inamounts from about 5% to about 94%, by weight of the final chewing gumcomposition, and preferably in amounts from about 15% to about 45%, andmore preferably in amounts from about 15% to about 35%, and mostpreferably in amounts from about 20% to about 30%, by weight of thefinal chewing gum composition.

The gum base composition may contain conventional elastomer solvents toaid in softening the elastomer base component. Such elastomer solventsmay comprise terpinene resins such as polymers of Alpha-pinene orβ-pinene, methyl, glycerol or pentaerythritol esters of rosins ormodified rosins and gums, such as hydrogented, dimerized or polymerizedrosins or mixtures thereof. Examples of elastomer solvents suitable foruse herein include the pentaerythritol ester of partially hydrogenatedwood or gum rosin, the pentaerythritol ester of wood or gum rosin, theglycerol ester of wood rosin, the glycerol ester of partially dimerizedwood or gum rosin, the glycerol ester of polymerized wood or gum rosin,the glycerol ester of tall oil rosin, the glycerol ester of wood or gumrosin and the partially hydrogenated wood or gum rosin and the partiallyhydrogenated methyl ester of wood or rosin, mixtures thereof, and thelike. The elastomer solvent may be employed in amounts from about 5% toabout 75%, by weight of the gum base, and preferably from about 45% toabout 70%, by weight of the gum base.

A variety of traditional ingredients may be included in the gum base ineffective amounts such as plasticizers or softeners such as lanolin,palmitic acid, oleic acid, stearic acid, sodium stearate, potassiumstearate, glyceryl triacetate, glyceryl lecithin, glyceryl monostearate,propylene glycol monostearate, acetylated monoglyceride, glycerine,mixtures thereof, and the like may also be incorporated into the gumbase to obtain a variety of desirable textures and consistencyproperties. Waxes, for example, natural and synthetic waxes,hydrogenated vegetable oils, petroleum waxes such as polyurethane waxes,polyethylene waxes, paraffin waxes, microcrystalline waxes, fatty waxes,sorbitan monostearate, tallow, propylene glycol, mixtures thereof, andthe like may also be incorporated into the gum base to obtain a varietyof desirable textures and consistency properties. These traditionaladditional materials are generally employed in amounts up to about 30%,by weight of the gum base, and preferably in amounts from about 3% toabout 20%, by weight of the gum base.

The gum base may include effective amounts of mineral adjuvants such ascalcium carbonate, magnesium carbonate, alumina, aluminum hydroxide,aluminum silicate, talc, tricalcium phosphate, dicalcium phosphate andthe like as well as mixtures thereof. These mineral adjuvants may serveas fillers and textural agents. These fillers or adjuvants may be usedin the gum base in various amounts. Preferably the amount of filler whenused will be present in an amount up to about 60%, by weight of thechewing gum base.

The chewing gum base may additionally include the conventional additivesof coloring agents, antioxidants, preservatives and the like. Forexample, titanium dioxide and other dyes suitable for food, drug andcosmetic applications, known as F.D.& C. dyes, may be utilized. Anantioxidant such as butylated hydroxytoluene (BHT), butylatedhydroxyanisole (BHA), propyl gallate, and mixtures thereof, may also beincluded. Other conventional chewing gum additives known to one havingordinary skill in the chewing gum art may also be used in the chewinggum base.

The gum composition may include effective amounts of conventionaladditives selected from the group consisting of sweetening agents(sweeteners), plasticizers, softeners, emulsifiers, waxes, fillers,bulking agents, mineral adjuvants, flavoring agents (flavors,flavorings), coloring agents (colorants, colorings), antioxidants,acidulants, thickeners, mixtures thereof and the like. Some of theseadditives may serve more than one purpose. For example, in sugarless gumcompositions, the sweetener, e.g., sorbitol or other sugar alcohol ormixtures thereof, may also function as a bulking agent. Similarly, insugar containing gum compositions, the sugar sweetener can also functionas a bulking agent.

The plasticizers, softeners, mineral adjuvants, colorants, waxes andantioxidants discussed above as being suitable for use in the gum basemay also be used in the gum composition. Examples of other conventionaladditives which may be used include emulsifiers, such as lecithin andglyceryl monostearate, thickeners, used alone or in combination withother softeners, such as methyl cellulose, alginates, carrageenan,xanthan gum, gelatin, carob, tragacanth, locust bean, and carboxy methylcellulose, acidulants such as malic acid, adipic acid, citric acid,tartaric acid, fumaric acid, and mixtures thereof, and fillers, such asthose discussed above under the category of mineral adjuvants. Thefillers when used may be utilized in an amount up to about 60%, byweight of the gum composition.

Bulking agents (carriers, extenders) suitable for use in chewing gumsinclude sweetening agents selected from the group consisting ofmonosaccharides, disaccharides, poly-saccharides, sugar alcohols, andmixtures thereof; polydextrose; maltodextrins; minerals, such as calciumcarbonate, talc, titanium dioxide, dicalcium phosphate, and the like.Bulking agents may be used in amounts up to about 90%, by weight of thefinal gum composition, with amounts from about 40% to about 70%, byweight of the gum composition being preferred, with from about 50% toabout 65%, by weight, being more preferred and from about 55% to about60%, by weight of the chewing gum composition, being most preferred.

The sweetening agent used may be selected from a wide range of materialsincluding water-soluble sweeteners, water-soluble artificial sweeteners,water-soluble sweeteners derived from naturally occurring water-solublesweeteners, dipeptide based sweeteners, and protein based sweeteners,including mixtures thereof. Without being limited to particularsweeteners, representative categories and examples include:

(a) water-soluble sweetening agents such as monosaccharides,disaccharides and polysaccharides such as xylose, ribulose, glucose(dextrose), mannose, galactose, fructose (levulose), sucrose (sugar),maltose, invert sugar (a mixture of fructose and glucose derived fromsucrose), partially hydrolyzed starch, corn syrup solid,dihydrochalcones, monellin, steviosides, glycyrrhizin, and sugaralcohols such as sorbitol, mannitol, maltitol, hydrogenated starchhydrolysates and mixtures thereof;

(b) water-soluble artificial sweeteners such as soluble saccharin salts,i.e., sodium or calcium saccharin salts, cyclamate salts, the sodium,ammonium or calcium salt of3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one-2,2-dioxide, the potassinmsalt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-one 2,2-dioxide(Acesulfame-K), the free acid form of saccharin, and the like;

(c) dipeptide based sweeteners, such as L-aspartic acid derivedsweeteners, such as L-aspartyl-L-phenylalanine methyl ester (Aspartame)and materials described in U.S. Pat. No. 3,492,131,L-Alpha-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alanin-amidehydrate (Alitame), methyl esters of L-aspartyl-L-phenylglyceine andL-aspartyl-L-2,5-dihydrophenyl-glycine,L-aspartyl-2,5-dihydro-L-phenylalanine;L-aspartyl-L-(1-cyclohexen)-alanine, and the like;

(d) water-soluble sweeteners derived from naturally occurringwater-soluble sweeteners, such as chlorinated derivatives of ordinarysugar (sucrose), known, for example, under the product designation ofSucralose; and

(e) protein based sweeteners such as thaumaoccous danielli (Thaumatin Iand II).

In general, an effective amount of sweetener is utilized to provide thelevel of bulk and/or sweetness desired, and this amount will vary withthe sweetener selected. This amount of sweetener will normally bepresent in amounts from about 0.0025% to about 90%, by weight of the gumcomposition, depending upon the sweetener used. The exact range ofamounts for each type of sweetener is well known in the art and is notthe subject of the present invention. The amount of sweetener ordinarilynecessary to achieve the desired level of sweetness is independent fromthe flavor level achieved from flavor oils.

Preferred sugar based-sweeteners are sugar (sucrose), corn syrup andmixtures thereof. Preferred sugarless sweeteners are the sugar alcohols,artificial sweeteners, dipeptide based sweeteners and mixtures thereof.Preferably, sugar alcohols are used in the sugarless compositionsbecause these sweeteners can be used in amounts which are sufficient toprovide bulk as well as the desired level of sweetness. Preferred sugaralcohols are selected from the group consisting of sorbitol, xylitol,maltitol, mannitol, and mixtures thereof. More preferably, sorbitol or amixture of sorbitol and mannitol is utilized. The gamma form of sorbitolis preferred. An artificial sweetener or dipeptide based sweetener ispreferably added to the gum compositions which contain sugar alcohols.

The coloring agents useful in the gum compositions are used in amountseffective to produce the desired color. These coloring agents includepigments which may be incorporated in amounts up to about 6% by weightof the gum composition. A preferred pigment, titanium dioxide, may beincorporated in amounts up to about 2%, and preferably less than about1% by weight of the composition. The colorants may also include naturalfood colors and dyes suitable for food, drug and cosmetic applications.These colorants are known as F.D.& C. dyes and lakes. The materialsacceptable for the foregoing uses are preferably water-soluble.Illustrative nonlimiting examples include the indigoid dye known asF.D.& C. Blue No.2, which is the disodium salt of5,5-indigotindisulfonic acid. Similarly, the dye known as F.D.& C. GreenNo.1 comprises a triphenylmethane dye and is the monosodium salt of 4-4-(N-ethyl -p-sulfoniumbenzylamino)diphenylmethylene!-1-(N-ethyl-N-p-sulfoniumbenzyl)-delta-2,5-cyclohexadieneimine!. A fullrecitation of all F.D.& C. colorants and their corresponding chemicalstructures may be found in the Kirk-Othmer Encyclopedia of ChemicalTechnology, 3rd Edition, in volume 5 at pages 857-884, which text isincorporated herein by reference.

Suitable oils and fats usable in gum compositions include partiallyhydogenated vegetable or animal fits, such as coconut oil, palm kerneloil, beef tallow, lard, and the like. These ingredients when used aregenerally present in amounts up to about 7%, by weight, and preferablyup to about 3.5%, by weight of the gum composition.

In accordance with this invention, therapeutically effective amounts ofthe therapeutic wound healing compositions of the present invention maybe admixed into a chewing gum. These amounts are readily determined bythose skilled in the without the need for undue experimentation. In apreferred embodiment, the final chewing gum composition will comprisethe therapeutic wound healing composition in an amount from about 0.1%to about 10% and a chewing gum composition in a quantity sufficient tobring the total amount of composition to 100%, by weight of the chewinggum composition. In a more preferred embodiment, the final chewing gumcomposition will comprise the therapeutic wound healing composition inan amount from about 0.1% to about 5%, and in a most preferredembodiment, the final chewing gum composition will comprise thetherapeutic wound healing composition in an amount from about 0.1% toabout 2%, and a chewing gum composition in a quantity sufficient tobring the total amount of composition to 100%, by weight of the chewinggum composition.

The present invention extends to methods of making the therapeuticchewing gum compositions. The therapeutic wound healing compositions maybe incorporated into an otherwise conventional chewing gum compositionusing standard techniques and equipment known to those skilled in theart. The apparatus useful in accordance with the present inventioncomprises mixing and heating apparatus well known in the chewing gummanufacturing arts, and therefore the selection of the specificapparatus will be apparent to the artisan.

For example, a gum base is heated to a temperature sufficiently highenough to soften the base without adversely effecting the physical andchemical make up of the base. The optimum temperatures utilized may varydepending upon the composition of the gum base used, but suchtemperatures are readily determined by those skilled in the art withoutundue experimentation.

The gum base is conventionally melted at temperatures that range fromabout 60° C. to about 120° C. for a period of time sufficient to renderthe base molten. For example, the gum base may be heated under theseconditions for a period of about thirty minutes just prior to beingadmixed incrementally with the remaining ingredients of the base such asthe plasticizer, fillers, the bulking agent and/or sweeteners, thesoftener and coloring agents to plasticize the blend as well as tomodulate the hardness, viscoelasticity and formability of the base. Thechewing gum base is then blended with the therapeutic wound healingcomposition of the present invention which may have been previouslyblended with other traditional ingredients. Mixing is continued until auniform mixture of gum composition is obtained. Thereafter the gumcomposition mixture may be formed into desirable chewing gum shapes.

In a specific embodiment, the invention is directed to a therapeuticpharmaceutical composition for preventing and reducing injury tomammalian cells, and increasing the resuscitation rate of injuredmammalian cells, which comprises:

(A) a therapeutically effective amount of a therapeutic wound healingcomposition of Embodiment One (I) selected from the group consisting of:

(I.A) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acid required for the repair of cellular membranesand resuscitation of mammalian cells;

(I.C) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D) (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be selected from the groupconsisting of pharmaceutical appliances, topical vehicles, andingestible vehicle.

In another specific embodiment, the invention is directed to a methodfor preparing a therapeutic pharmaceutical composition for preventingand reducing injury to mammalian cells, and increasing the resuscitationrate of injured mammalian cells, which comprises the steps of:

(A) providing a therapeutically effective amount of a therapeutic woundhealing composition of Embodiment One (I.A-D) selected from the groupconsisting of:

(I.A) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.B) (a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and rcsuscitation of mammalian cells;

(I.C) (a) an antioxidant; and

(b) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(I.D) (a) lactate selected from the group consisting of lactic acid,pharmaceutically acceptable salts of lactic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) providing a pharmaceutically acceptable carrier, and

(C) admixing the therapeutic wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form atherapeutic pharmaceutical composition.

Throughout this application, various publications have been referenced.The disclosures in these publications are incorporated herein byreference in order to more fully describe the state of the art.

The present invention is further illustrated by the following exampleswhich are not intended to limit the effective scope of the claims. Allparts and percentages in the examples and throughout the specificationand claims are by weight of the final composition unless other, wisespecified.

E. Examples Of The Therapeutic Wound Healing Compositions Of EmbodimentOne (I.A-D)

Study 1

This study demonstrates a comparison of the viability of U937 monocyticcells after exposure of the cells to various antioxidants andcombinations of antioxidants. This study also demonstrate a comparisonof the levels of hydrogen peroxide produced by U937 monocytic cells andmammalian epidermal keratinocytes after exposure of the cells to variousantioxidants and combinations of antioxidants. The results of this studyare illustrated in FIGS. 1-4 and examples 1-26 below.

Mammalian epidermal keratinocytes and monocytes were employed to examinethe ability of various antioxidants to reduce levels of hydrogenperoxide in these cells. Hydrogen peroxide was measured after the cellswere exposed to ultraviolet light in the wavelength range from 290 to320 nm (UV-B) or to the inflammatory compound12-0-tetradecanoyl-phorbol-13-acetate (TPA). Sodium pyruvate was testedat various concentrations to determine the effect of concentrations ofthis antioxidant on the hydrogen peroxide production by epidermal cellsand monocytes. Magnesium pyruvate, calcium pyruvate, zinc pyruvate, andcombinations of sodium pyruvate with ascorbic acid, lactic acid, andVitamin E were then tested to determine the effect of these salts andcombinations of antioxidants on the hydrogen peroxide production byepidermal cells and monocytes.

Mammalian epidermal keratinocytes were isolated by trypsinization ofepithelial sheets and grown in modified basal MCDB 153 mediumsupplemented with epidermal growth factor, bovine pituitary extract, andhydrocortisone. Cells were maintained in a humidified incubator with 5%carbon dioxide at 37° C. Keratinocytes were seeded in 60 mm culturedishes at a cell density of 3×10⁵ cells per dish and the cultures wereexposed to 1 M.E.D. dose of ultraviolet-B light (100 mJ/cm²) or treatedwith 100 ng/ml of TPA.

U937 monocytic cells are a cultured cell line grown in RPMI media with10% fetal calf serum. Cells were maintained in a 60 mm culture dish at5% carbon dioxide at 37° C. at a seeding density not exceeding 1×10⁶cells per dish.

Sodium pyruvate, lactic acid, ascorbic acid, and Vitamin E weredissolved in distilled water, with sufficient surfactant. Theconcentrations of the sodium pyruvate solutions prepared were 1 mM, 10mM, 50 mM, 100 mM, and 200 mM. The concentrations of the lactic acidsolutions prepared were 1.0%, 0.1%, and 0.05%. The concentrations of theascorbic acid solutions prepared were 1.0%, 0.1%, 0.05%, and 0.025%. Theconcentrations of the Vitamin E solutions prepared were 1 U, 10 U, 50 U,and 100 U. The test solutions were adjusted to a pH value of 7.4 with1.0N sodium hydroxide solution and then sterile filtered. Theappropriate concentration of test solution or combination of testsolutions was added to the cells immediately prior to exposure of thecells to ultraviolet light-B or TPA 100ng/ml!. Stock solutions wereprepared so that the vehicle did not constitute more than 1% of thetotal volume of the culture media.

Inncellular hydrogen peroxide production by mammalian epidermalkeratinocytes and U937 monocytes was measured using dichlorofluoresceindiacetate (DCFH-DA, Molecular Probes, Eugene, Oreg.). DCFH-DA is anon-polar non-fluorescent compound that readily diffuses into cellswhere it is hydrolyzed to the polar non-fluorescent derivative DCFHwhich then becomes trapped within the cells. In the presence ofinncellular hydrogen peroxide, DCFH is oxidized to the highlyfluorescent compound DCF. Hence, cellular fluorescence intensity isdirectly proportional to the level of intracellular hydrogen peroxideproduced. Cellular fluorescence intensity can be monitored byfluorimetry and by flow cytometry.

Mammalian epidermal keratinocytes and U937 cultured monocytes (1×10⁶ perdish) were incubated at 37° C. with 5 uM of DCFH-DA. Production ofhydrogen peroxide was measured using a Coulter Profile analytical flowcytometer. Linear and log intensity of green fluorescence data wascollected. For each analysis, a quantity of 10,000 to 20,000 events wasaccumulated. Optical alignment for the instrument was performed daily.Coefficients of variation for forward angle light scatter and integratedgreen fluorescence were generally less than two. Each analysis wasrepeated three times and the quantitation of fluorescence was expressedin terms of femtomoles (fmol, 10⁻¹⁵ moles) of DCF oxidized per cell,which is a direct measure of the intracellular hydrogen peroxideproduced. Alternatively, in the saturated and unsaturated fatty acidexamples in examples 27-52, fluorimetry was used to assess the DCFoxidation per cell.

The viability of the U937 monocytic cells after exposure of the cells tovarious antioxidants for 24 hours was measured. The viability of thecells was determined by exposing the cells to the dye propidium iodide.Permeable cell membranes which absorbed the dye were not consideredviable. The viability of the cells was represented as the percentage ofcells that excluded propidium iodide. FIG. 1 depicts in bar graph formatthe viability of U937 monocytic cells after exposure of the cells to noantioxidant (Example 1, control), to sodium pyruvate (Example 2), toascorbic acid (Example 3), to lactic acid (Example 4), and to Vitamin E(Example 5). FIG. 2 depicts in bar graph format the viability of U937monocytic cells af exposure of the cells to various combinations ofantioxidants. Specifically, the viability of U937 monocytic cells wasmeasured after exposure to no antioxidant (Example 6, control), toascorbic acid and lactic acid (Example 7), to ascorbic acid and VitaminE (Example 8), to sodium pyruvate and ascorbic acid (Example 9), tosodium pyruvate and lactic acid (Example 10), to sodium pyruvate andVitamin E (Example 11), to lactic acid and Vitamin E (Example 12), andto sodium pyruvate, ascorbic acid, and lactic acid (Example 13).

FIG. 1 shows that ascorbic acid is cytotoxic to monocytes atconcentrations as low as 0.25%. FIG. 2 shows that the cytotoxicity ofascorbic acid was reversed by the addition of 10 mM of sodium pyruvate.FIGS. 1 and 2 show that the viability rate of 15% to 20% of the cellswhen treated with ascorbic acid was increased to 95% to 98% uponaddition of sodium pyruvate. Lactic acid and Vitamin E did not reversethe cytotoxicity of ascorbic acid.

Sodium pyruvate was then tested at various concentrations to determinethe effect of concentrations of this antioxidant on the hydrogenperoxide production by epidermal cells and monocytes. Mammalianepidermal keratinocytes and monocytes were exposed to (a) 1 M.E.D. doseof ultraviolet light-B and (b) 100 ng/ml of12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of sodiumpyruvate at the following concentrations: 200 mM, 100 mM, 50 mM, 10 mM,1 mM.

The optimum concentration of sodium pyruvate to reduce the hydrogenperoxide production by epidermal cells and monocytes was found to be 10mM. Concentrations of sodium pyruvate of 50 mM and above were cytotoxicto both epidermal keratinocytes and monocytes.

Magnesium pyruvate, calcium pyruvate, zinc pyruvate, ascorbic acid,lactic acid, and Vitamin E, and combinations of sodium pyruvate withascorbic acid, lactic acid, and Vitamin E were then tested to determinethe effect of these salts and combinations of antioxidants on thehydrogen peroxide production by epidermal cells and monocytes. Thefollowing test solutions were prepared.

(a) sodium pyruvate 10 mM!;

(b) zinc salt 10 mM!;

(c) magnesium salt 10 mM!;

(d) calcium salt 10 mM!;

(e) sodium pyruvate 10 mM! and ascorbic acid 0.025%!;

(f) sodium pyruvate 10 mM! and lactic acid 0.05%!;

(g) sodium pyruvate 10 mM!, lactic acid, 0.05%!, and ascorbic acid0.025%!;

(h) lactic acid 1.0%, 0.1%, and 0.05%!;

(i) ascorbic acid 1.0%, 0.1%, 0.05%, and 0.025%!;

(j) Vitamin E 1 U, 10 U, 50 U, and 100 U!; and

(k) vehicle solvent controls.

There was no significant difference among the zinc, magnesium, andcalcium salts of pyruvic acid on the hydrogen peroxide production byepidermal cells and monocytes. The zinc and calcium salts of pyruvicacid induced differentiation of keratinocytes. For convenience, thesodium salt was used in subsequent tests.

The optimum concentration of lactic acid to reduce the hydrogen peroxideproduction by epidermal cells and monocytes was found to be 0.05%. Theoptimum concentration of ascorbic acid was found to be 0.025%. Thehigher concentrations of both of these compounds were found to becytotoxic to both types of cells. The optimum concentration of Vitamin Ewas found to be 50 U.

FIG. 3 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells after exposure of the cells to noantioxidant (Example 14, control), to sodium pyruvate (Example 15), toascorbic acid (Example 16), to lactic acid (Example 17), and to VitaminE (Example 18). Sodium pyruvate and Vitamin E significantly reduced thehydrogen peroxide production by monocytes.

FIG. 4 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells after exposure of the cells to variouscombinations of antioxidants. Specifically, the levels of hydrogenperoxide produced by U937 monocytic cells were measured after exposureto no antioxidant (Example 19, control), to ascorbic acid and lacticacid (Example 20), to ascorbic acid and Vitamin E (Example 21), tosodium pyruvate and ascorbic acid (Example 22), to sodium pyruvate andlactic acid (Example 23), to sodium pyruvate and Vitamin E (Example 24),to lactic acid and Vitamin E (Example 25), and to sodium pyruvate,ascorbic acid, and lactic acid (Example 26). The combination of lacticacid (0.05%) and Vitamin E (50 U) significantly reduced the hydrogenperoxide production by monocytes.

The morphological alterations in epidermal keratinocytes were observedin control cultures and in cultures exposed to ultraviolet-B. Cells inthe layer closest to the dermis are basal keratinocytes. These cellsproliferate and migrate into the spinous and granular layers of theepidermis where the cells begin to differentiate. The differentiationpattern results in cells enucleating and forming cornified envelopes atthe uppermost portion of the epidermis, the statum corneum. Thedifferentiation of keratinocytes is controlled by the levels of calcium,magnesium, and other elements in the medium. Cells in culture systemspromoting differentiation appear as an epidermal sheet formingattachments or tight junctions with each other. Keratinocytes thatbecome nonadherent or float in the media were considered responding to acytotoxic event.

The following morphological alterations in the mammalian epidermalkeratinocytes were observed for the following control cultures:

10 mM Sodium Pyruvate:

Tight junctions of cells were formed and the proliferation rate of thecells was higher than the rate of the control cells.

0.025% Ascorbic Acid:

Cells were floating in a cytotoxic response to ascorbic acid.

0.025% Ascorbic acid and 10 mM Sodium Pyruvate:

Few tight junctions of cells were observed and cells appeared similar tothe cells in the sodium pyruvate culture.

0.05% Lactic Acid:

Cells appeared dramatically altered as an epidermal sheet and as flatgranular cells.

0.05% Lactic Acid and 10 mM Sodium Pyruvate:

Cells formed an epidermal sheet but appeared smaller than the cell inthe lactic acid culture.

50 U Vitamin E:

Cells appeared the same as the cells in the control culture.

50 U Vitamin E and 10 mM Sodium Pyruvate:

Cells increased in number and changed in appearance resembling the cellsin the sodium pyruvate culture.

The following morphological alterations in the mammalian epidermalkeratinocytes were observed for the corresponding cultures exposed toultraviolet light-B, 100 mJoules, for 24 hours:

10 mM Sodium Pyruvate:

Cells proliferated more rapidly than the cells in the control culture.

0.025% Ascorbic Acid:

Cells were nonadherent and floating in a cytotoxic response to ascorbicacid greater than the cytotoxic response of the corresponding cellswithout ultraviolet-B light exposure.

0.05% Lactic Acid:

Cells formed an epidermal sheet and were more granular than cells in thecontrol culture without uitraviolet-B light exposure.

50 U Vitamin E:

Cell growth was inhibited but cells appeared similar to cells in thecontrol culture without ultraviolet-B light exposure.

50 U Vitamin E and 10 mM Sodium Pyruvate:

Cells appeared similar to cells in the control culture and proliferatedto a greater extent than cells in the control cultures withoutultraviolet-B light exposure.

Morphological alterations in the U937 monocytic cell line were alsoobserved for control cultures and cultures exposed to ultravioletlight-B, 100 mJoules, for 24 hours. The following compounds andcombination of compounds, at the concentrations set out below,significantly inhibited the levels of hydrogen peroxide produced by U937monocytic cells

Sodium pyruvate at 10 mM and 50 mM;

Vitamin E at 50 U and 100 U; and

Lactic acid at 0.05% and Vitamin E at 50 U.

Examples Of The Therapeutic Wound Healing Compositions Of Embodiment One(I.A-D)

Study 2

This study demonstrates a comparison of the levels of hydrogen peroxideproduced by U937 monocytic cells and epidermal keratinocytes afterexposure of the cells to various combinations of antioxidants with andwithout a mixture of saturated and unsaturated fatty acids. The resultsof this study are illustrated in FIGS. 5-7 and examples 27-52 below.

Mammalian epidermal keratinocytes and U937 monocytic cells and the testsolutions of sodium pyruvate, lactic acid, ascorbic acid, and Vitamin Ewere prepared as describe above for Examples 1-26. Intracellularhydrogen peroxide production by the mammalian epidermal keratinocytesand U937 monocytes was also measured as described above.

A mixture of fatty acids derived from chicken fat was prepared foraddition to the cultured cells by mixing 0.1% of the chicken fat withthe culture media. At the temperature of the culture media, 37° C., thechicken fat was miscible. This chicken fat mixture was added to culturesof cells prior to exposure of the cells to ultraviolet-B light or TPAtreatment.

As set out in examples 1-26, mammalian epidermal keratinocytes andmonocytes were exposed to (a) 1 M.E.D. dose of ultraviolet light-B and(b) 100 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in the presence ofvarious antioxidants and combinations of antioxidants with and without amixture of saturated and unsaturated fatty acids 0.1%, 0.5%, and 1.0%chicken fat!.

FIG. 5 depicts in bar graph format the levels of hydrogen peroxideproduced by U937 monocytic cells exposure of the cells to variouscombinations of antioxidants with and without a mixture of saturated andunsaturated fatty acids. Specifically, the levels of hydrogen peroxideproduced by U937 monocytic cells were measured after exposure to lacticacid and Vitamin E without fatty adds (Example 27) and with fatty acids(Example 28), to ascorbic acid and lactic acid without fatty acids(Example 29) and with fatty acids (Example 30), and to ascorbic acid andVitamin E without fatty acids (Example 31) and with fatty acids (Example32). The ability of the combinations of lactic acid and Vitamin E,ascorbic acid and lactic acid, and ascorbic acid and Vitamin E to reducethe hydrogen peroxide production by monocytes was increased in thepresence of fatty acids. The most effective combination to reduce thehydrogen peroxide production of monocytes was lactic acid (0.05%) andVitamin E (50 E) in the presence of a mixture of saturated andunsaturated fatty acids (0.5%).

FIG. 6 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes after exposure of the cells tovarious antioxidants with and without a mixture of saturated andunsaturated fatty acids. Specifically, the levels of hydrogen peroxideproduced by epidermal keratinocytes were measured after exposure to noantioxidant without fatty acid (Example 33, control) and with fatty acid(Example 34), to sodium pyruvate without fatty acid (Example 35) andwith fatty acids (Example 36), to ascorbic acid without fatty acid(Example 37) and with fatty acid (Example 38), to lactic acid withoutfatty acid (Example 39) and with fatty acids (Example 40), and toVitamin E without fatty acids (Example 41) and with fatty acid (Example42). The ability of sodium pyruvate and Vitamin E to reduce the hydrogenperoxide production by epidermal keratinocytes was increased in thepresence of fatty acids. The most effective combinations to reduce thehydrogen peroxide production of epidermal keratinocytes were sodiumpyruvate in combination with a mixture saturated and unsaturated fattyacid and Vitamin E in combination with a mixture of saturated andunsaturated fatty acid.

FIG. 7 depicts in bar graph format the levels of hydrogen peroxideproduced by epidermal keratinocytes after exposure of the cells tovarious combinations of antioxidants with and without a mixture ofsaturated and unsaturated fatty acids. Specifically, the levels ofhydrogen peroxide produced by epidermal keratinocytes were measuredafter exposure to no antioxidant without fatty acids (Example 43,control) and with fatty acids (Example 44), to sodium pyruvate andascorbic acid without fatty acids (Example 45) and with fatty acids(Example 46), to sodium pyruvate and lactic acid without fatty acids(Example 47) and with fatty acids (Example 48), to sodium pyruvate andVitamin E without fatty acids (Example 49) and with fatty acids (Example50), and to ascorbic acid and Vitamin E without fatty acids (Example 51)and with fatty acids (Example 52). The ability of all combinations ofantioxidants to reduce the hydrogen peroxide production by epidermalkeratinocytes was increased in the presence of fatty acids. In order ofpotency, the most effective combinations to reduce the hydrogen peroxideproduction of epidermal keratinocytes were sodium pyruvate and VitaminE, sodium pyruvate and lactic acid, and Vitamin E, each in combinationwith a mixture of saturated and unsaturated fatty acids (0.5%).

Because of the cytotoxicity of cells towards ascorbic acid describedabove, the ascorbic acid combinations without sodium pyruvate were notconsidered significantly different from the control test solution.

Summary Analysis Of The Data From Studies 1 and 2

Human epidermal keratinocytes were isolated by trypsinization ofepithelial sheets and grown in modified base MCDB 153 mediumsupplemented with epidermal growth factor and bovine pituitary extract.Cells were seeded in culture dishes at a density of 3×10⁵ /dish. Priorto exposure to UV B light (100 mJ/cm²) or treatment with 100 ng/ml TPA,the cultures were treated with the appropriate concentration of woundhealing components. Intracellular production of hydrogen peroxide wasmeasured using DCFH-DA, a nonpolar compound that readily diffuses intocells, hydrolyzed to a nonpolar derivative. In the presence ofintracellular hydrogen peroxide, DCFH is oxidized to a highlyfluorescent compound DCF. Thus, cellular fluorescence intensity isdirectly proportional to levels of hydrogen peroxide produced and can bemonitored by flow cytometry. Hydrogen peroxide is cytotoxic, thereforelower levels of hydrogen peroxide production is desirable for cellularviability.

In all cases, the three component wound healing composition surpassedthe predicted outcomes, clearly demonstrating unpredicted synergy.

    ______________________________________                                        Results                                                                       1                      2      3       4                                       ______________________________________                                        1 -   Control          250    250     0                                       2 -   Fatty Acids      250    230     -20                                           (0.5%)                                                                  3 -   Sodium Pyruvate  250    490     +240                                          (10 mM)                                                                 4 -   Vitamin E        250    400     +150                                          (50 units)                                                              5 -   Pyruvate &       250    430     +180                                          Fatty Acids                                                             6 -   Vitamin E &      250    200     -50                                           Fatty Acids                                                             7 -   Pyruvate &       250    290     +40                                           Vitamin E                                                               8 -   Pymvate &        250    120     -130                                          Vitamin E & Fatty Acids                                                 ______________________________________                                         Column 1 shows the different treatment groups.                                Column 2 shows the producfion of H.sub.2 O.sub.2 in control cells             (fmol/cell).                                                                  Column 3 shows the production of H.sub.2 O.sub.2 after treatment with         wound healing components.                                                     Column 4 shows the difference in production of H.sub.2 O.sub.2 from           control after the treatment.                                             

All comparisons were assessed against the controls, which produced 250H₂ O₂ fmol/cell. The positive numbers represent H₂ O₂ production inexcess of the control and the negative numbers represent H₂ O₂production below the control. These results are set out in FIG. 8.

Combination of Single Ingredient Effects

Fatty Acids (-20) & Vitamin E (+150) & Pyruvate (+240)

+370 Is The Predicted Three Component Effect

-130 Is The Wound healing composition Actual Effect

500 Is The Difference Between Predicted Effect minus Actual effect

(Synergy)

Combination of Paired and Single Ingredients

Pyruvate & Fatty Acid (+180) & Vitamin E (+150)

+330 Is The Predicted Three Component Effect

-130 Is The Wound healing composition Actual Effect

460 Is The Difference between Predicted Effect minus Actual Effect

(Synergy)

Vitamin E & Fatty Acids (-50) & Pyruvate (+240)

+190 Is The Predicted Three Component Effect

-130 Is The Wound healing composition Actual Effect

320 Is The Difference between Predicted Effect minus Actual Effect

(Synergy)

Pyruvate & Vitamin E (+40) & Fatty Acids (-20)

+20 Is The Predicted Three Component Effect

-130 Is The Wound healing composition Actual Effect

150 Is The Difference between Predicted Effect minus Actual Effect

(Synergy)

In all cases, the three component wound healing composition surpassedthe predicted outcomes clearly demonstrating unpredicted synergy.

Examples Of The Therapeutic Wound Healing Compositions Of Embodiment One(I.A-D)

Study 3

This study demonstrates a comparison of the wound healing abilities ofthe therapeutic wound healing compositions of the present inventionversus conventional wound healing compositions. The results of thisstudy are illustrated in examples A-D.

The wound healing compositions of Examples A-D were prepared having thecompositions set out in Table A.

    ______________________________________                                                   Examples                                                                        A                                                                Ingredient   Prep-H ™                                                                            B         C     D                                       ______________________________________                                        sodium pyruvate                                                                            --       2%        --    --                                      vitamin E    --       1%        --    --                                      chicken fat  --       2%        --    --                                      LYCD         2000 U*  2400 U    2400 U                                                                              --                                      shark liver oil                                                                            3%       3%        3%    --                                      petrolatum   in       64%       66.5% 68%                                     mineral oil  amounts  22.53%    25.03%                                                                              26.8%                                   paraffin     totaling 5%        5%    5%                                      emulsifier   100%     0.2%      0.2%  0.2%                                    ______________________________________                                         *These components are present in Preparation H                           

Wound healing composition A was commercially available Preparation H™.Wound healing composition B was a petrolatum base formulation containinglive yeast cell derivative, shark oil, and a mixture of sodium pyruvate,vitamin E, and chicken fat. Wound healing composition C was a petrolatumbase formulation containing live yeast cell derivative and shark oil.Wound healing composition D was a petrolatum base formulation only.

Wound healing studies were carried out using hairless mice (SKR-1,Charles River) 6-8 weeks in age. One group of mice were untreated as acontrol group and were referred to as Example E. In each group therewere 6 mice for evaluation at either day 3 or day 7 for a total numberof 60 animals in the study. The mice were anesthetized with ether and amidline 3 cm full thickness longitudinal incision was made with a number10 scalpel blade. Incisions were closed using steel clips at 1 cmintervals. Formulations A-D set out above were applied in a randomizedblinded study to the wounds on day 0 at 2 hours following wounding andreapplied at 24 hour intervals during the 7 days of the study. Thewounds were examined daily and scored on a basis of 0-5 for closure oneach day of the study, with a score of 5 representing the wound besthealed.

The animals were sacrificed on day 3 and day 7 using cervicaldislocation. The dorsal skin including the incision was dissectedwithout the subcutaneous tissue. The skin was placed in neutral bufferedformalin and subsequently sectioned and stained with hematoxylin andeosin. The wounds were examined microscopically and representativetissue sections were photographed.

On each day of the experiment, the score and rank order of theformulations for closure of wounds and speed of healing were as follows:

    B(5)>>D(4)>>C(2)>/=E, Control(2)>A                         (1)

Photographs of the wounded mice on day 4 are set out in FIGS. 9A-9D and10.

FIGS. 9A-9D and 10 show that Formulation B, which was a petrolatum baseformulation containing live yeast cell derivative, shark off, and amixture of sodium pyruvate, vitamin E, and chicken fat, was asignificantly better wound healing agent than the other formulations.These results are supported by the subjective grading of the woundclosures and the speed of healing on each day (1-7) of the experiment aswell as on the objective histological examination of tissue sections tomeasure the extent of inflammatory cell infiltrate within the wound andthe extent of epithelialization at the wound edges. The final result wasthat less sear tissue was present at day 7 on the mice treated withFormulation B.

Formulation D, which was a white petrolatum formulation only, was judgedto be significantly more effective to promote healing than eitherFormulation C, which was a petrolatum base formulation containing sharkliver oil and live yeast cell derivative, or Formulation A, which wasPreparation H™. The superior ability of Formulation D over Formulation Cto improve healing may result from a delay in the healing process causedwhen the live yeast cell derivative is depleted and the cells shift toan alternative nutrient source. The presence of the mixture of sodiumpyruvate, vitamin E, and chicken fat in Formulation B apparently offsetsthe depletion of the live yeast cell derivative.

Formulation C, which was a petrolatum base formulation containing liveyeast cell derivative and shark oil, was judged comparable to thecontrol (untreated wound) in speed of wound closure and extent ofhealing. Formulation A, which was Preparation H™, appeared to be theleast effective healing formulation by both subjective grading of woundhealing and by objective examination of tissue sections. The superiorability of Formulation D and Formulation C over Formulation A to improvehealing may be due to their ability to act as an occlusive wounddressing that prevents transepidermal water loss and thus promoteshealing and wound closure. The poor ability of Formulation A to improvehealing may be due to the potential cytotoxicity of phenylmercuricnitrate present in Preparation H™ as a preservative.

These results show that the wound healing compositions of the presentinvention which comprise a mixture of sodium pyruvate, vitamin E, andchicken fat increase the proliferation and resuscitation rate ofmammalian cells. The wound healing compositions mediate low levels ofoxygen in the initial stages of healing to suppress oxidative damage andhigher levels of oxygen in the later stages of healing to promotecollagen formation.

II. SUNSCREEN WOUND HEALING COMPOSITIONS

A. Embodiment Two (I.A-D+X)

Applicant has discovered therapeutic sunscreen-wound healingcompositions (I.A-D+X) which comprise a therapeutically effective amountof a sunscreen agent and an anti-inflammatory agent (collectivelyreferred to as X) and the wound healing compositions of Embodiment One(I.A-D). Preferably, the wound healing composition (I.A) comprises (a)pyruvate, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids. Sunscreen agents can help prevent sunburn byscreening ultra violet light but do not heal injured mammalian cells.Anti-inflammatory agents can reduce inflammation (erythema) in a patientbut do not promote the wound healing process. Wound healing compositionscan increase the resuscitation rate of injured mammalian cells and theproliferation rate of new mammalian cells to replace dead cells. Woundhealing compositions can also minimize oxygen radical damage from ultraviolet light. Applicants have found that the combination of a sunscreenagent, an anti-inflammatory agent, and a wound healing compositionresults in a therapeutic sunscreen-wound healing compositions useful forminimizing and treating sunburn damage. The sunscreen-wound healingcompositions may optionally contain a therapeutically effective amountof a topical anesthetic to further reduce the duration and severity ofsunburn.

The combination of the sunscreen agent, the anti-inflammatory agent, andthe wound healing compositions of the present invention provides apharmaceutical composition useful for minimizing and treating sunburndamage and having an enhanced ability to prevent and reduce injury tomammalian cells and further increase the resuscitation rate of injuredmammalian cells. The tissue damage associated with sunburn is believedto be caused by the production of cellular produced active oxygenspecies. Combination of the sunscreen agent, anti-inflammatory agent,and the wound healing compositions helps suppress such reactiveoxygen-linked tissue injury.

Sunscreen agents are compounds which provide broad spectrum protectionfrom ultra violet A and ultra violet B light from the sun. The sunscreenagents in the sunscreen-wound healing compositions of the presentinvention may be selected from a wide range of therapeutic agents andmixtures of therapeutic agents. Nonlimiting illustrative specificexamples of sunscreen agents include ethylhexyl p-methoxycinnamate,octyl methoxycinnamate, octyl dimethyl p-aminobenzoic acid, 2-ethylhexylsalicylate, octyl salicylate, menthyl anthranilate, octocrylene,padimate o, titanium dioxide, urea, and oxybenzone. Preferably, thesunscrcen agent is oxybenzone.

The amount of sunscreen agent used in the present invention is atherapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particular sunscreenagent. In general, the amount of sunscreen agent present is the ordinarydosage required to obtain the desired result. Such dosages are known tothe skilled practitioner in the medical arts and are not a part of thepresent invention. In a preferred embodiment, the sunscreen agent in thesunscreen-wound healing composition is present in an amount from about1% to about 30%, preferably from about 2% to about 25%, and morepreferably from about 4% to about 20%, by weight.

Anti-inflammatory agents are compounds that counteract or suppress theinflammatory process. The anti-inflammatory agents in thesunscreen-wound healing compositions of the present invention may beselected from a wide variety of steroidal, non-steroidal, and salicylatewater-soluble and water-insoluble drugs and their acid addition ormetallic salts. Both organic and inorganic salts may be used providedthe anti-inflammatory agent maintains its medicament value. Theanti-inflammatory agents may be selected from a wide range oftherapeutic agents and mixtures of therapeutic agents which may beadministered in sustained release or prolonged action form. Nonlimitingillustrative specific examples of non-steroidal anti-inflammatory agentsinclude the following medicaments: ibuprofen, naproxen, sulindac,diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium,fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolactromethamine, diclofenac, and evening primrose oil (containing about 72%linoleic acid and about 9% gamma-linolenic acid). Nonlimitingillustrative specific examples of salicylate anti-inflammatory agentsinclude the following medicaments: acetylsalicylic acid, mesalamine,salsalate, diflunisal, salicylsalicylic acid, and choline magnesiumtrisalicylate. Nonlimiting illustrative specific examples of steroidalanti-inflammatory agents include the following medicaments: flunisolide,triamcinoline, triamcinoline acetonide, beclomethasone diproprionate,betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone,predinisone, methyl prednisolone, and prodnisolone.

Preferred anti-inflammatory agents to be employed may be selected fromthe group consisting of ibuprofen, naproxen, sulindac, diflunisal,piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoprobencalcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine,diclofenac, evening primrose oil, acetylsalicylic acid, mesalamine,salsalate, diflunisal, salicylsalicylic acid, choline magnesiumtrisalicylate, flunisolide, triamcinoline, triamcinoline acetonide,beclomethasone diproprionate, betamethasone diproprionate,hydrocortisone, cortisone, dexamethasone, predinisone, methylprednisolone, and prednisolone. In a preferred embodiment, theanti-inflammatory agent is selected from the group consisting ofibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin,etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen,mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, andevening primrose oil. In a more preferred embodiment, theanti-inflammatory agent is evening primrose oil.

The anti-inflammatory agent of the present invention may be used in manydistinct physical forms well known in the pharmaceutical art to providean initial dosage of the anti-inflammatory agent and/or a furthertime-release form of the anti-inflammatory agent. Without being limitedthereto, such physical forms include free forms and encapsulated forms,and mixtures thereof.

The amount of anti-inflammatory agent used in the present invention is atherapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particularanti-inflammatory agent. In general, the amount of anti-inflammatoryagent present is the ordinary dosage required to obtain the desiredresult. Such dosages are known to the skilled practitioner in themedical arts and are not a part of the present invention. In a preferredembodiment, the anti-inflammatory agent in the sunscreen-wound healingcomposition is present in an amount from about 0.01% to about 10%,preferably from about 0.1% to about 5%, and more preferably from about1% to about 3%, by weight.

In yet another preferred embodiment, the therapeutic sunscreen-woundhealing compositions of the present invention further comprise a topicalanesthetic agent. Anesthetic agents are compounds that induce the lossof tactile sensibility and the sensation of pain. The anesthetic agentsin the sunscreen-wound healing compositions of the present invention maybe selected from a wide range of therapeutic agents and mixtures oftherapeutic agents. Nonlimiting illustrative specific examples oftopical anesthetic agents include pramoxine hydrochloride, lidocaine,and benzocaine.

The amount of anesthetic agent used in the present invention is atherapeutically effective amount and may vary depending upon thetherapeutic dosage recommended or permitted for the particularanesthetic agents. In general, the amount of anesthetic agents presentis the ordinary dosage required to obtain the desired result. Suchdosages are known to the skilled practitioner in the medical arts andare not a pan of the present invention. In a preferred embodiment, theanesthetic agent in the sunscreen-wound healing composition is presentin an amount from about 1% to about 30%, preferably from about 2% toabout 25%, and more preferably from about 2.5% to about 20%, by weight.

B. Methods For Making The Sunscreen-Wound Healing Compositions OfEmbodiment Two (I.A-D+X)

The present invention extend to methods for making the therapeuticsunscreen-wound healing compositions (I.A-D+X). In general, atherapeutic sunscreen-wound healing composition is made by forming anadmixture of the wound healing components of Embodiment One (I.A-D), asunscreen agent, and an anti-inflammatory agent. In a first aspect ofEmbodiment Two (I.A+X), a sunscreen-wound healing therapeuticcomposition is made by forming an admixture of a sunscreen agent, ananti-inflammatory agent, and a wound healing composition comprising (a)a pyruvate, (b) an antioxidant, and (c) a mixture of saturated andunsaturated fatty acids. In a second aspect of Embodiment Two (I.B+X), asunscreen-wound healing therapeutic composition is made by forming anadmixture of a sunscreen agent, an anti-inflammatory agent, and a woundhealing composition comprising (a) a pyruvate, (b) a lactate, and (c) amixture of saturated and unsaturated fatty acids. In a third aspect ofEmbodiment Two (I.C+X), a sunscreen-wound healing therapeuticcomposition is made by forming an admixture of a sunscreen agent, ananti-inflammatory agent, and a wound healing composition comprising (a)an antioxidant, and (b) a mixture of saturated and unsaturated fattyacid. In a fourth aspect of Embodiment Two (I.D+X), a sunscreen-woundhealing therapeutic composition is made by forming an admixture ofadmixture of a sunscreen agent, an anti-inflammatory agent, and a woundhealing composition comprising (a) a lactatc, (b) an antioxidant, and(c) a mixture of saturated and unsaturated fatty acid.

In a preferred embodiment, the invention is directed to a method forpreparing a therapeutic sunscreen-wound healing composition (I.A+X)useful to minimize and treat sunburn damage which comprises the steps ofadmixing a therapeutically effective amount of the followingingredients:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacid are those fatty acid required for the repair of cellular membranesand resuscitation of mammalian cells.

C. Methods For Employing The Sunscreen-Wound Healing Compositions OfEmbodiment Two (I.A-D+X)

The present invention extend to methods for employing the therapeuticsunscreen-wound healing compositions (I.A-D+X). In general, atherapeutic composition is employed by contacting the therapeuticcomposition with the skin to be exposed to the sun. In a preferredembodiment, the invention is directed to a method for minimizing andtreating sunburn in a human with a sunscreen-wound healing composition(I.A+X) which comprises the steps of:

(A) providing a therapeutically effective amount of a sunscreen-woundhealing composition which comprises:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acid required for the repair of cellular membranesand resuscitation of an cells; and

(B) contacting the sunscreen-wound healing composition with the skin ofthe human prior to exposure to the sun.

D. Augmented Sunscreen-Wound Healing Compositions Of Embodiment Two(I.A-D+X+M)

In another aspect of Embodiment Two, the therapeutic sunscreen-woundhealing compositions (I.A-D+X) of the present invention may be furthercombinedwith medicaments useful for treating wounds (M) to formaugmented sunscreen-wound healing compositions (I.A-D+X+M). In thisembodiment, the combination of the sunscreen-wound healing compositionof the present invention and the medicament useful for treating woundprovides an augmented sunscreen-wound healing composition having anenhanced ability to increase the proliferation and resuscitation rate ofmammalian cells. For example, the therapeutic compositions of thepresent invention may be used in combination with medicaments useful fortreating wounds such as immunostimulating agents (Betafectin™),antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, tretinoin, other sunscreen agents, dermatologicalagents, topical antihistamine agents, antibacterial agents, bioadhesiveagents, respiratory bursting inhibitors (lactic acid, adenosine),inhibitors of prostaglandin synthesis (ibuprofen, aspirin, indomethacin,meclofenomic acid, retinoic acid, padimate O, meclomen, oxybenzone),steroidal anti-inflammatory agents (corticosteroid including syntheticanalogs), antimicrobial agents (neosporin ointment, silvadine),antiseptic agents, anesthetic agents (pramoxine hydrochloride,lidocaine, benzocaine), cell nutrient media, burn relief medications,sun burn medicatious, acne preparations, insect bite and stingmedications, wound cleansers, wound dressings, scar reducing agents(vitamin E), and the like, and mixtures thereof, to further enhance theproliferation and resuscitation rate of mammalian cells. Preferably, themedicament useful for treating wounds is selected from the groupconsisting of immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,tretinoin, sunscreen agents, dermatological agents, topicalantihistamine agents, antibacterial agents, bioadhesive agents,respiratory bursting inhibitors, inhibitors of prostaglandin synthesis,antimicrobial agents, cell nutrient media, scar reducing agents, andmixtures thereof. More preferably, the medicament useful for treatingwounds is selected from the group consisting of immunostimulatingagents, antiviral agents, antikeratolytic agents, anti-inflammatoryagents, antifungal agents, acne treating agents, sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, and mixtures thereof.

In a preferred embodiment, the invention is directed to an augmentedsunscreen-wound healing composition (I.A+X+M) useful to minimize andtreat sunburn damage which comprises:

(A) a therapeutic sunscreen-wound healing composition which comprises atherapeutically effective amount of:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition, wherein the wound healing compositioncomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacid are those fatty acid required for the repair of cellular membranesand resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

The present invention extend to methods for making the augmentedsunscreen-wound healing compositions. In general, the augmentedcompositions are made by admixing the therapeutic sunscreen-woundhealing composition with the medicament useful for treating wounds toprepare the augmented sunscreen-wound healing composition.

The present invention also extend to methods for employing the augmentedsunscreen-wound healing compositions. In general, an augmentedsunscreen-wound healing composition is employed by contacting thecomposition with the skin to be exposed to the sun. In a specfficembodiment, the invention is directed to a method for minimizing andtreating sunburn in a human with an augmented sunscreen-wound healingcomposition (I.A+X+M) which comprises the steps of:

(A) providing a therapeutically effective amount of a sunscreen-woundhealing composition which comprises:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition, wherein the wound healing compositioncomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acid wherein the fattyacid are those fatty acid required for the repair of cellular membranesand resuscitation of mammalian cells; and

(B) a medicament useful for treating wounds.

(C) contacting the augmented sunscreen-wound healing composition withthe skin of the human prior to exposure to the sun.

The types of wounds which may be healed using the sunscreen-woundhealing compositions and the augmented sunscreen-wound healingcompositions of the present invention are inflammation wounds induced bysunburn. The therapeutic compositions may be used topically to protectand accelerate the healing of injured tissue.

Methods for treating sunburn comprise topically administering thecompositions of the present invention directly to the skin of the hunanprior to exposure to the sun. The composition is maintained in contactwith the skin for a period of time sufficient to increase theproliferation and resuscitation rate of the cells.

E. Formulations Of The Sunscreen-Wound Healing Compositions OfEmbodiment Two (I.A-D+X) and (I.A-D+X+M)

Once prepared, the inventive therapeutic sunscreen-wound healingcompositions and augmented sunscreen-wound healing compositions may bestored for future use or may be formulated in effective amounts withpharmaceutically acceptable carriers such as pharmaceutical appliancesand topical vehicles to prepare a wide variety of pharmaceuticalcompositions. The pharmaceutically acceptable carriers which may beemployed and the methods used to prepare the pharmaceutical compositionshave been described above in connection with the formulations of thewound healing compositions of Embodiment One (I.A-D).

In a preferred embodiment, the invention is directed to asunscreen-wound healing pharmaceutical composition which comprises:

(A) a therapeutic sunscreen-wound healing composition (I.A+X) whichcomprises:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition, wherein the wound healing compositioncomprises:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells; and

(B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.

In another preferred embodiment, the invention is directed to a methodfor preparing a pharmaceutical composition for increasing theproliferation and resuscitation rate of mammalian cells, which comprisesthe steps of:

(A) providing a therapeutically effective amount of a sunscreen-woundhealing composition (I.A+X) which comprises:

(1) a sunscreen agent;

(2) an anti-inflammatory agent; and

(3) a wound healing composition comprising:

(a) pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;

(b) an antioxidant; and

(c) a mixture of saturated and unsaturated fatty acids wherein the fattyacids are those fatty acids required for the repair of cellularmembranes and resuscitation of mammalian cells;

(B) providing a pharmaceutically acceptable carrier, and

(C) admixing the sunscreen-wound healing composition from step (A) andthe pharmaceutically acceptable carrier from step (B) to form apharmaceutical composition.

F. Examples Of The Sunscreen-Wound Healing Compositions Of EmbodimentTwo (I.A-D+X)

Study 1

SUMMARY

This study was conducted to determine the potential of formulations withmoisatrizers and antioxidants to effectively prevent or reduce solarsimulator derived erythema in normal volunteers, and whether theerythema once manifested would be ameliorated with the test product. Theconcept was to develop a product that could protect and heal skin fromUV damage without the use of UV sunscreen blockers, i.e., PABA,oxybenzone, etc. Such protection would be extremely beneficial for coldsores and psoriatic lesions since UV exposure is known to exacerbatethese skin diseases.

Two products, a lotion and a cream, were evaluated for their sunprotection potential (Phase I) and for product efficacy in the treatmentof solar simulator induced erythema (Phase II). Ten subjects completedPhase I of the study. Thirteen subjects completed Phase II of the study.An 8% Homosalate solution served as a control in Phase I.

Under the conditions employed in this study, both the cream and lotiontest products provided low level sun protection (SPF values less then 2)and minor improvements in UV-induced erythema as compared withirradiated control sites not treated with test products.

INTRODUCTION

Objective

This study was conducted to determine the potential for two woundhealing compositions containing formulations for preventing solarsimulator derived erythema in normal volunteers and whether, theerythema once manifested, would be ameliorated with the test woundhealing composition product.

Rationale

The demands for sun screen and skin care products make up the fastestgrowing cosmetics sector in the United States. The growth in sun careproducts is continuing because overexposure to the sun is believed toproduce many ill-effects on the skin. It is known that even high sunprotectant factor (SPF-15) products do not fully protect human skinagainst cumulative skin damage. The nature of this damage is believed tobe associated with the presence of various "reactive" oxygen species. Aproduct to reduce the levels of photogenerated reactive oxygen speciesshould provide meaningful long term skin benefits. The purpose of thisexperiment was to determine whether a formula which containsmoisturizers and antioxidants could effectively eliminate or reduce thedamage produced from overexposure to UV light. The objective was also todetermine if applying the product before and/or af UV damage wouldprovide beneficial effects to the damaged area.

BACKGROUND

Two (2) products were submitted for testing in this two phase study.Each study group consisted of ten (10) volunteer subjects. The productswere considered reasonably safe for testing on human subjects. Theprotocol followed for Phase I was that established by the OTC panel andpublished in the Federal Register, Aug. 25, 1978 (Vol 43, No. 166).

MATERIALS AND METHODS

A sample of each test material(s) was reserved and stored for a periodof five (5) years. At the conclusion of the clinical study, theremaining test material(s) was discarded. All information regarding thereceipt, storage and disposition of the material(s) was also recorded ona Clinical Material Record form. All test materials were kept in alocked product storage room accessible to clinical staff members only.

    ______________________________________                                        Test Materials                                                                ______________________________________                                        Product (1) Identification:                                                                         Lotion                                                  Description:          White Lotion                                            Quantity Provided:    1 × 6 oz.                                         Amount applied:       0.1 g/50 cm.sup.2                                       Product (2) Identification:                                                                         Cream                                                   Description:          Yellow Cream                                            Quantity Provided:    1 × 4 oz.                                         Amount applied:       0.1 g/50 cm.sup.2                                       ______________________________________                                    

Product (1) was Lubriderm™ Lotion containin 2% sodium pyruvate, 1%vitamin E, and 1% chicken fat. Product (2) was Lubriderm™ Creamcontaining aquaphor/petrolatum cream with 10% sodium pyruvate, 5%vitamin E, and 5% chicken fat.

EXPERIMENTAL DESIGN

Inclusion Data

Individuals included in the study were eighteen (18) years of age orolder; were fair and had uniformly-colored skin on the lower thoracicarea of the back which would allow a discernible erythema; were free ofany systemic or dermatologic disorder which would interfere with thetest results; had completed a phototesting Medical Screening form and aMedical/Personal History form; and had read, understood, and signed aninformed consent agreement.

Exclusion Data

Individuals excluded in the study were individuals who had any visibleskin disease at the test site which would have interfered with the testresults; were receiving systemic or topical drugs or medication such asantihistamines or anti-inflammatorics which might have interfered withthe test results; were taking medication suspected of causingphotobiological reactions (i.e., tetracyclins, thiazides, etc.); hadactive atopic dermatiitis or eczema; had psoriasis; were currently undertreatment for asthma; had cataracts; had a history of skin cancer; werepregnant or planning a pregnancy or nursing a child; or has a knownsensitivity to cosmetics, skin care products, or topical drugs asrelated to the products being tested.

Light Source

A Xenon Arc Solar Simulator (150 W) was used which has a continuousemission spectrum in the UVA and UVB range (290 to 400 nanometers). Lessthan 1% of its total energy was composed of "non-solar" wavelengthsbelow 290 mm. The output was monitored daily, using the Robert-Bergemeter. Additional measurements of the energy output were taken andrecorded as needed.

Definitions of Terms in Clinical Evaluation

MED, Minimal Erythema Dose

The time of light exposure necessary to produce a minimal perceptibleerythema (redness) on the skin, discernible sixteen (16) to twenty four(24) hours later.

SPF, Sun Protection Factor

The ultraviolet energy required to produce an MED on protected skin,divided by the ultraviolet energy required to produce an MED onunprotected skin.

PCD, Product Category Designation

1. Minimal Sun Protection Product

Sunscreen products that provide an SPF value of 2 to 4, and offer theleast protection from sunburning, but permit suntanning.

2. Moderate Sun Protection Product

Sunscreen products that provide an SPF value of 4 to 6, and offermoderate protection from sunburning, and permit some suntaning.

3. Extra Sun Protection Product

Sunscreen products that provide an SPF value of 6 to 8, and offer extraprotection from sunburning, and permit limited suntanning.

4. Maximal Sun Protection Product

Sunscreen products that provide an SPF value of 8 to under 15, and offermaximal protection from sunburning, and permit little or no suntanning.

5. Ultra Sun Protection Product

Sunscreen products that provide an SPF value of 15 or greater, and offerthe most protection from sunburning and permit no suntanning.

Phase I--Determination of Minimal Erythemal Dose (MED)

MED is defined as the time of light exposure required to produce aminimal perceptible erythema reaction discernible sixteen (16) totwenty-four (24) hours after irradiation using a standardizedultraviolet light source that emits UVB(290-320 nm) as all or part ofits emission spectrum. Subjects who were candidates for this testinghave skin types commonly referred to as Fitzpatrick skin types, ofCategory I, II, or III according to the following definitions:

Category I Always burns easily, never tans.

Category II Always burns easily, tans minimally.

Category III Burns moderately, tans gradually (light brown).

Category IV Burns minimally, always tans well (moderate brown).

Category V Rarely burns, tans very well (moderate brown).

Category VI Never burns, deeply pigmented.

An area, other than the test site, was drawn with a marking pen on thesubject's back and divided into five (5) equal sites. The anticipatedMED was estimated from skin type (I, II, or III) of the individual andthe irradiation size calculated based on energy output of the Xenonlamp. The (5) sites were irradiated on the basis of a geometricprogression of 1.25, i.e., each site receiving 25% more exposure thanthe site to its left. Sixteen (16) to twenty-four (24) hours later, theMED was determined by establishing the site which exhibited the leastamount of perceptible erythema.

SPF Determination

The test material was applied as follows:

A 50 cm² area was marked on the back of the subject. Then, a uniformapplication of 0.1g or ml or product was applied to the area withcareful spreading and gentle but thorough robbing over the entire markedarea. The product was permitted to dry for fifteen (15) to thirty (30)minutes.

The MED previously determined on untreated skin was multiplied by theexpected SPF of the product. For example, for an MED of twenty-five (25)seconds, and a test product whose SPF is estimated at two (2), themedian exposure time for this product would be 2×25 seconds=50 seconds.

The irradiation for site exposure was again calculated on the basis of ageometric progression of 1.25 with the site to the right receiving moreirradiation than the site to its left. The sites were then exposed toultraviolet light from the solar simulator. The procedure was repeatedusing the control product, an 8% homosalate standard.

In addition, an area of untreated skin was exposed to ultraviolet lightfrom the solar simulator to again determine the MED of unprotected skin.Sixteen (16) to twenty-four (24) hours later, the MED for the treatedand untreated skin was determined and the SPF calculated.

Following exposure, all immediate responses were noted for reddening,vesiculation, tanning, darkening of skin, etc.

Study Flow Chart

Day

1 Obtained informed consent; completed medical screening form; conductedUV irradiation for MED determination.

2 Determined MED; calculated UV exposure times; irradiated test productsite, control product site, and untreated sites for second MEDdetermination.

3 Evaluated all sites; calculated SPF.

To assure the uniform evaluation of sunscreen products, a standardsunscreen was used concomitantly in the test procedure, specifically instep 2, irradiated control product site. This control product was aneight percent (8%) homosalate preparation.

Rejection of Test Data

There were three (3) reasons for rejection of test data.

1. Sometimes the exposure either failed to elicit an MED response on thetreated or unprotected skin sites or elicited responses at all five (5)irradiated sites. In either event, that test was considered a technicalfailure and had to be discarded. If the subject reacted to one or moreexposures on the unprotected control site, but not on the treated site,then a minimal estimate could be obtained. However, this estimate wouldnot be used in assessing the Mean of the SPF values.

2. The response on the treated sites was randomly absent, whichindicated the product was not spread evenly. Therefore, no assessment ofprotection was possible.

3. If the SPF values obtained experimentally were well outside theexpected range, the values were discarded.

Phase II--Treatment of Erythema

Ten subjects whose MEDs were determined by the previous method (Phase I)had twelve test site areas delineated on the back: five sites wereexposed to one times the MED; five sites were exposed to two times theMED, and two sites were not exposed to UV light. The cream test productwas applied (0.1 g/50 cm²) and rubbed into two of the 1×MED sites, twoof the 2×MED sites, and to one of the unexposed sites. The lotion testproduct was applied to two separate 1×MED sites, two separate 1×MEDsites, and one separate unexposed site. The two remaining exposed areas(one 1×MED and one 2×MED) served as controls and did not have productapplied. All sites were covered, but not occluded, with a gauzedressing. Application of the product to the same sites was repeatedapproximately six hours later and the following mornings and afternoonsfor four consecutive days. Evaluation of the test sites was made eachafternoon prior to product application by a trained clinical evaluatorwho had no participation in any other aspect of the study procedure. Afinal evaluation was made on the morning of Day 8, the last productapplication having occurred on the previous Friday (Day 4). Evaluationsof each test site were made according to the following scale:

0.0--minimal or doubtful erythema

0.5--faint erythema

1.0--distinct, but mild erythema

2.0--moderate erythema

RESULTS AND DISCUSSION

Phase I

Two (2) products, a lotion and a cream; were submitted for evaluation oftheir sun protective potential. A total of ten (10) subjects between theages of 25 and 54 were enrolled and completed the evaluation of the testproducts and the homosalate control.

A low level of sun protection was observed; an SPF value of 1.9±0.2(N=9) was obtained for the cream and a value of 1.7±0.3 (N=8) wasobtained for the lotion.

Phase II

The same two (2) products were also evaluated for their product efficacyin the treatment of solar-simulator induced erythema. A total of fifteen(15) subjects between the ages of 20 and 66 were enrolled and thirteen(13) subjects completed the study. Two (2) subjects discontinued forpersonal reasons.

Product treated sites exposed to two MEDs of irradiation exhibited minorimprovements in resolution of erythema as compared to the irradiatednon-product treated sites. Comparisons between erythema levels at Day 2and subsequent evaluation days was used as the basis of analysis. Day 2was selected as the baseline day since increases in erythema from Day 1to Day 2 occurred frequently but appeared to have stabilized by Day 2.Comparison of erythema results on Days 3, 4, 5 and 8 to Day 2 suggest aminor acceleration in the relation of erythema responses. Specifically,in 12 instances among the 52 comparisons (23%), thirteen (13) subjectswith 4 comparisons each, comparing Day 2 to Days 3, 4, 5, and 8,reductions in erythema associated with both the cream and the lotionwere manifested.

While it is noted with caution that both the proportion and degree ofimprovanent associated with the product test sites were minor, it isalso noteworthy that there was only one instance out of the total of onehundred four (104) comparisons for the two products in which the controlsite showed greater degree of improvement than the test site.Furthermore, this one improvement occurred only for the Day 8/Day 2comparison.

The cream product showed improvement over the control site in twosubjects for all four comparison intervals, in one subject at two of thefour intervals, and two other subjects at one of the four intervals.Similarly, the lotion product showed improvement in one subject at allfour intervals, in one subject at three intervals, in two subjects attwo intervals, and, in one subject at one interval. Conversely, eight ofthirteen subjects showed no differences at any of the four intervals incomparisons between the test product sites and the control sites forboth the cream and the lotion products. While the effects noted heremust again be stressed as minimal, it is, nonetheless, noteworthy thatimprovements, where noted, favored effects of the test products over thecontrol and not the opposite: twenty-four instances of possible positiveeffects from the two test products compared to one such possibleinstance at the control site.

A similar analysis of data associated with the 1×MED sites did notprovide the same results due to the low level and variable erythematousresponses observed. This is believed to be due to the fact that minorperturbations of ultra violet lamp output are likely to be sufficient toresult in sub-erythema and mildly erythema responses even on the samesubject at adjoining sites.

In conclusion, under the conditions employed in this clinical study,both the cream and lotion test products provided low-level sunprotection and indications of minor improvement in UV-induced erythemaas compared with irradiated control sites not treated with testproducts. However, it is clear that these effects are, at best, minorand that verification of efficacy would require a larger proportion ofstudy subjects. Formulation changes (vehicle, level of wound healingcomposition) or product application (e.g., multiple applications orocclusive conditions) may also be worth exploring.

I claim:
 1. A therapeutic sunscreen-wound healing oomposition useful tominimize and treat sunburn damage which comprises a therapeuticallyeffective amount of:(1) a sunscreen agent; (2) an anti-inflammatoryagent; and (3) a wound healing composition, wherein the wound healingcomposition comprises:(a) pyruvate selected from the group consisting ofpyruvic acid, pharmaceutically acceptable salts of pyruvic acid, andmixtures thereof; (b) an antioxidant; and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the repair of cellular membranes resuscitation of injuredmammalian cells; wherein components a, b, and c are present in amountssufficient to synergisticaly enhance wound healing.
 2. The compositionaccording to claim 1, wherein the sunscreen agent is selected from thegroup consisting of ethylhexyl p-methoxycinnamate, octylmethoxycinnamate, octyl dimethyl p-aminobenzoic acid, 2-ethylhexylsalicylate, octyl salicylate, menthyl anthranilate, octocrylene,padimate o, titanium dioxide, urea, and oxybenzone.
 3. The compositionaccording to claim 2, wherein the sunscreen agent is oxybenzone.
 4. Thecomposition according to claim 1, wherein the anti-inflammatory agent isselected from the group consisting of ibuprofen, naproxen, sulindac,diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium,fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolactromethamine, diclofenac, evening primrose oil, acetylsalicylic acid,mesalamine, salsalate, diflunisal, salicylsalicylic acid, cholinemagnesium trisalicylate, flunisolide, triamcinoline, triamcinolineacetonide, beclomethasone diproprionate, betamethasone diproprionate,hydrocortisone, cortisone, dexamethasone, predinisone, methylprednisolone, and prednisolone.
 5. The composition according to claim 4,wherein the anti-inflammatory agent is evening primrose oil.
 6. Thecomposition according to claim 1, wherein the pyruvate is selected fromthe group consisting of pyruvic acid, lithium pyruvate, sodium pyruvate,potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate,manganese pyruvate, methyl pyruvate, α-ketoglutaric acid,pharmaceutically acceptable salts of pyruvic acid, prodrugs of pyruvicacid, and mixtures thereof.
 7. The composition according to claim 6,wherein the pyruvate is sodium pyruvate.
 8. The composition according toclaim 1, wherein the antioxidant is selected from the group consistingof all forms of Vitamin A; all forms of carotene; all forms of VitaminC; all forms of Vitamin E; Vitamin E esters which readily undergohydrolysis to Vitamin E; prodrugs of Vitamin A, carotene, Vitamin C, andVitamin E; pharmaceutically acceptable salts of Vitamin A, carotene,Vitamin C, and Vitamin E, and mixtures thereof.
 9. The compositionaccording to claim 8, wherein the antioxidant is Vitamin E acetate. 10.The composition according to claim 1, wherein the mixture of saturatedand unsaturated fatty acids is selected from the group consisting ofanimal and vegetable fats and waxes.
 11. The composition according toclaim 10, wherein the mixture of saturated and unsaturated fatty acidsis selected from the group consisting of human fat, chicken fat, cowfat, sheep fat, horse fat, pig fat, and whale fat.
 12. The compositionaccording to claim 11, wherein the mixture of saturated and unsaturatedfatty acid comprises lauric acid, myristic acid, myristoleic acid,pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid,margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid,arachidic acid, and gadoleic acid.
 13. The composition according toclaim 1, wherein the sunscreen agent is present in the therapeutic woundhealing composition in an amount from about 1% to about 30%, by weightof the therapeutic wound healing composition.
 14. The compositionaccording to claim 1, wherein the anti-inflammatory agent is present inthe therapeutic wound healing composition in an amount from about 0.01%to about 10%, by weight of the therapeutic wound healing composition.15. The composition according to claim 1, wherein pyruvate is present inthe therapeutic wound healing composition in an amount from about 10% toabout 50%, by weight of the therapeutic wound healing composition. 16.The composition according to claim 1, wherein the antioxidant is presentin the therapeutic wound healing composition in an amount from about0.1% to about 40%, by weight of the therapeutic wound healingcomposition.
 17. The composition according to claim 1, wherein themixture of saturated and unsaturated fatty acids is present in thetherapeutic wound healing composition in an amount from about 10% toabout 50%, by weight of the therapeutic wound healing composition. 18.The composition according to claim 1, further comprising atherapeutically effective amount of a topical anesthetic.
 19. A methodfor minimizing and treating sunburn in a human with a sunscreen-woundhealing composition which comprises administering to a mammal in needthereof:a therapeutically effective amount of a sunscreen-wound healingcomposition which comprises:(1) a sunscreen agent; (2) ananti-inflammatory agent; and (3) a wound healing compositioncomprising:(a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof; (b) an antioxidant; and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the resuscitation of injured mammalian cells; whereincomponents a, b, and c are present in synergistic amounts sufficient tosynergistically enhance wound healing.
 20. The method according to claim19, further comprising a therapeutically effective amount of a topicalanesthetic.
 21. The method according to claim 20, further comprising atherapeutically effective amount of a topical anesthetic.
 22. Anaugmented sunscreen-wound healing composition useful to minimize andtreat sunburn damage which comprises:(A) a therapeutic sunscreen-woundhealing composition which comprises a therapeutically effective amountof:(1) a sunscreen agent; (2) an anti-inflammatory agent; and (3) awound healing composition, wherein the wound healing compositioncomprises:(a) pyruvate selected from the group consisting of pyruvicacid, pharmaceutically acceptable salts of pyruvic acid, and mixturesthereof; (b) an antioxidant; and (c) a mixture of saturated andunsaturated fatty acids wherein the fatty acids are those fatty acidsrequired for the rapair of cellular membranes and resuscitation ofmammalian cells; wherein components a, b, and c are present in amountssufficient to synergistically enhance wound healing; and, (B) amedicament useful for treating wounds.
 23. The composition according toclaim 22, further comprising a therapeutically effective amount of atopical anesthetic.
 24. The augmented antiacne-wound healing compositionaccording to claim 22, wherein the medicament useful for treating woundsis selected from the group consisting of immunestimulating agents,antiviral agents, antikeratolytic agents, anti-inflammatory agents,antifungal agents, acne treating agents, other sunscreen agents,dermatological agents, antihistamine agents, antibacterial agents,bioadhesive agents, respiratory bursting inhibitors, of prostaglandinsynthesis, antimicrobial agents, antiseptic agents, anesthetic agents,cell nutrient media, burn relief medications, sun burn medications,insect bite and sting medications, wound cleansers, wound dressings,scar reducing agents, and mixtures thereof.
 25. A method for minimizingand treating sunburn in a human with an augmented sunscreen-woundhealing composition which comprises (the steps of:) administering to amammal in need thereof:((A) providing a therapeutically effective amountof a sunscreen-wound healing composition which comprises:(1) a sunscreenagent; (2) an anti-inflammatory agent; and(3) a wound healingcomposition, wherein the wound healing composition comprises: (a)pyruvate selected from the group consisting of pyruvic acid,pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;(b) an antioxidant; and (c) a mixture of saturated and unsaturated fattyacids wherein the fatty acids are those fatty acids required for therepair of cellular membranes and resuscitation of mammalian cells;wherein components a, b, and c are present in amounts sufficient tosynergistically enhance wound healing; and (3) a medicament useful fortreating wounds.
 26. The method according to claim 25, furthercomprising a therapeutically effective amount of a topical anesthetic.27. A sunscreen-wound healing composition which comprises:(A) atherapeutic sunscreen-wound healing composition which comprises:(1) asunscreen agent; (2) an anti-inflammatory agent; and (3) a wound healingcomposition which comprises:(a) pyruvate selected from the groupconsisting of pyruvic acid, pharmaceutically acceptable salts of pyruvicacid, and mixtures thereof; (b) an antioxidant; and (c) a mixture ofsaturated and unsaturated fatty acids wherein the fatty acids are thosefatty acids required for the repair of cellular membranes andresuscitation of mammalian cells; wherein components a, b, and c arepresent in amounts sufficient to synergistically enhance wound healing;and (B) a pharmaceutically acceptable carrier selected from the groupconsisting of pharmaceutical appliances, bioadhesives, and occlusivevehicles.
 28. The composition according to claim 26, further comprisinga therapeutically effective amount of a topical anesthetic.